Jefferson Duarte – VectorLabs

Quantum Dots

Vector Laboratories R&D — Fri, 24 Jan 2025 20:09:39 +0000

Quantum Dots

Posted in: Tips and Tricks

Utilizing dPEG® Products in Quantum Dots Applications

What are Quantum Dots?

Quantum dots are nanocrystals less than 10nm in diameter comprised of a semiconductor alloy core and coated with a shell of a different alloy metal such as Ag, Cd, Hg, Ln, P, Pb, Se, Te, and Zn ^[1]. Depending on their size and composition, quantum dots fluoresce at different wavelengths. The smaller the quantum dot, the more blue-shifted its emission wavelength while larger quantum dots (6 nm and larger) shift to red and near-IR. Quantum dots fluorescence makes them a suitable alternative to organic dyes in specific situations. These nanoparticles provide benefits including broad excitation spectra (absorb energy at a wide range of wavelengths), narrow emission spectra (emit a specific wavelength), and they do not suffer from photobleaching (degradation due to light intensity/oxidizing agents) ^[2].

What is dPEG?

The term “dPEG®” is Vector Laboratories’ trademarked acronym for “discrete polyethylene glycol” or “discrete PEG“. The “discrete” portion of the dPEG trademark indicates single molecular weight PEG technology and can help simplify product analysis. Comparatively, traditional PEGs are not single compounds.

Like traditional PEGs, our products contain an amphiphilic backbone of repeating ethylene oxide units. The term “amphiphilic” means that the compound is soluble in both water (or aqueous buffer) and organic solvents. All PEG products that do not contain hydrophobic substituents are soluble in water and in a variety of organic solvents. The addition of hydrophobic groups to PEG reduces the water solubility of some PEG products.

As part of Vector Laboratories’ BioDesign™ portfolio, dPEG was developed and continues to be manufactured using our proprietary synthetic and purification processes, ensuring lot-to-lot consistency and optimal performance.

Quantum Dot Synthesis

Synthesis of quantum dots involves injecting solutions of the semiconductor metals into hot (>300°C) organic solvent, such as octadecene, and allowing the metals to nucleate (the first step of crystallization) and form alloys. For emission wavelengths of 470-720nm CdSe, CdTe, and InP alloys are used, while PbS and PbSe are utilized for emission wavelengths >900nm.

Water solubility is one of the major hurdles that must be overcome when producing quantum dots for biological applications. When the quantum dots have reached their desired size, the surface is coated so that they can be easily isolated. Originally this was done using tri-n-octylphosphine oxide (TOPO) which results in a very hydrophobic quantum dot (Figure 1).

Figure 1: TOPO coated green quantum dot (approximately 3 nm diameter).

At this point, the TOPO must be replaced with either a water-soluble group or a more hydrophilic group, such as dPEG. The TOPO is commonly replaced by a thiol containing linker such as 2-mercaptoacetic acid, lipoic acid (also known as thioctic acid), 2-mercaptoethylamine (2-MEA), or cystamine (Figure 2).

thioctic acid, also known as lipoic acid
Chemical Formula: C₈H₁₄O₂S₂
Molecular Weight: 206.33

cyctamine, also known as
2,2’-disulfanediyldiethanamine
Chemical Formula: C₄H₁₂N₂S₄
Molecular Weight: 152.28

2-mercaptoacetic acid
Chemical Formula: C₂H₁₁NO₂S₂
Molecular Weight: 169.27

2-mercaptoethylamine (MEA)
Chemical Formula: C₂H₇NS
Molecular Weight: 77.15

Figure 2: Commonly used sulfur-containing compounds for attachment to gold surfaces such as quantum dots.

While these modifications allow for increased hydrophilicity, the water solubility can be increased even more by adding a dPEG to the acid or amine end by a simple coupling using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Alternatively, a thiol-dPEG could also be used directly on the surface of the quantum dot. Figure 3 shows dPEG compounds attached to a quantum dot either directly or coupled to a thiol monomer already on the surface.

Figure 3: Red Quantum Dot (approximately 6 nm diameter) with Attached dPEG compounds m-dPEG®₈-lipoamide (PN10800), m-dPEG®₈-acid (NHS ester) (PN10324, PN10260 – coupled to cystamine) and m-dPEG®₈-amine (PN10278 – coupled to mercaptoacetic acid)

Reducing Quantum Dot Non-Specific Binding with dPEG

Elizabeth L. Bentzen, et al., studied the effects of PEG on nonspecific binding associated with quantum dots and found that PEG as small as PEG350 (approximately dPEG₈), when coated over the surface of quantum dots, can reduce nonspecific binding in certain cells ^[3]. The results showed that, in the cells studied, the PEG length could possibly be shortened to 12 units without increasing the nonspecific binding. Using a dPEG in place of a traditional polydispersed PEG gives the advantages of lot-to-lot reproducibility and confidence in the identification of the final product.

Conclusion

dPEG has the potential to play an important role in quantum dots applications by addressing challenges such as water solubility and nonspecific binding. By leveraging Vector Laboratories’ proprietary dPEG products, researchers and developers can unlock new potential in quantum dots fluorescence and further expand their applications in research and medicine. For more information about dPEG products, click here.

Products

Browse our metal surface modification dPEG reagents here. For the products referenced in the caption to Figure 2, above, click on the links below:

PN10800, m-dPEG^®₈-lipoamide

PN10324, m-dPEG^®₈-acid

PN10260, m-dPEG^®₈-NHS ester

PN10278, m-dPEG^®₈-amine

References:

Kesrevani, R. K., & Sharma, A. K., Nanoarchitectured Biomaterials: Present Status and Future Prospects in Drug Delivery., William Andrew Publishing (2016), 35-66. [ScienceDirect]
Abramowitz, M., & Davidson, M. W. (n.d.), Overview of fluorescence excitation and emission fundamentals., Olympus Microscopy Resource Center, [OlympusConfocal]
Bentzen EL, Tomlinson ID, Mason J, Gresch P, Warnement MR, Wright D, Sanders-Bush E, Blakely R, Rosenthal SJ. Surface modification to reduce nonspecific binding of quantum dots in live cell assays. Bioconjug Chem., (2005), 16(6), 1488-94. [PubMed]
Hermanson, G. T. (2013). Bioconjugate Techniques, (3rd ed., pp. 455–463). Academic Press.

January 24, 2025

SEARCH THE BLOG

Quantum Dots

Vector Laboratories R&D

Click Chemistry Crosslinking with dPEG®

Vector Laboratories R&D

Maleimide Crosslinker Selection Guide

Vector Laboratories R&D

It Takes Two to Tango, Part 1: Bioconjugation

Gowtham SP

It Takes Two to Tango, Part 2: Applications of Bioconjugation

Gowtham SP

The post Quantum Dots appeared first on VectorLabs.

Click Chemistry Crosslinking with dPEG®

Vector Laboratories R&D — Mon, 23 Dec 2024 19:17:18 +0000

Click Chemistry Crosslinking with dPEG®

Posted in: Tips and Tricks

Click Chemistry Crosslinking with dPEG®

BioDesign™, by Vector Laboratories, provides extensive crosslinker offerings for use in applications where precision is required. One of these offerings is our proprietary dPEG® linkers, which are synthesized from high purity building blocks in a series of stepwise reactions to provide specific molecular weights, organic moieties, functional groups, and architectures. These products are part of our click chemistry crosslinking reagents and are well suited for a variety of uses.

What is dPEG?

Vector Laboratories developed and continues to manufacture these monodisperse PEG products using our proprietary synthetic and purification processes.

What is click chemistry?

Barry Sharpless coined the term click chemistry in 1998 ^[1]. He specified that these reactions are:

high yielding
stereospecific
modular
wide in scope

According to Sharpless, click chemistry reactions must “generate only inoffensive byproducts that can be removed by nonchromatographic methods….” Moreover, such reactions must be simple to perform with readily available starting materials and reagents. Additionally, they must be able to be conducted in benign solvents, such as water, or easily removable solvents. The resulting product “must be stable under physiological conditions” ^[1]. Click chemistry has become enormously important in chemical synthesis since Sharpless’s original paper. In 2022, the Nobel Prize in Chemistry was awarded to Carolyn R. Bertozzi, Morten Meldal, and K. Barry Sharpless for their work on click chemistry and bioorthogonal chemistry.

Why use click chemistry?

Numerous compounds and macromolecular constructs can be synthesized via click chemistry, thus solving many synthetic chemistry problems. These products include organic and organometallic nanoparticles, dendrimers, small molecule drugs, peptide drugs, and antibody-drug conjugates (ADCs).

Click chemistry qualifies as environmentally friendly “green chemistry” due to its use of benign or easily removable solvents ^[2]. Additionally, typical click chemistry reactions run faster in water than in organic solvents ^[1]. Research and experience demonstrate that click chemistry can be a highly useful synthetic process for reactions that require aqueous environments, such as cell-based assays ^[3]. Unfortunately, some types of click chemistry – for example, copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) – require cytotoxic metal salts. However, strain-promoted azide-alkyne cycloaddition (SPAAC), avoids these harmful salts.

Click chemistry crosslinking ideas using dPEG reagents.

Numerous click chemistry crosslinking products are available in the BioDesign portfolio, including azido-dPEG^®₁₁-amine (QBD-10524) (Figure 1). This dPEG reagent has an azide group and a primary amine residing on opposite ends of a single molecular weight PEG linker (molecular weight of 570.67 Daltons; linker length of 36 atoms, ≈42.7 Å).

Figure 1: Azido-dPEG®₁₁-amine: a BioDesign click chemistry crosslinking reagent.

This product crosslinks carboxylic acids with alkyne-containing compounds. The amine end of the molecule forms amide bonds with carboxyl groups directly using a carbodiimide such as EDC. More commonly, the amide bond forms by reacting the amine with an active ester such as an N-hydroxysuccinimidyl (NHS) or 2,3,5,6-tetrafluorophenyl (TFP) ester. On the opposite end of the linker, the azide group reacts with alkyne-containing molecules via click chemistry reactions such as CuAAC or SPAAC. Please see Scheme 1 below.

Scheme 1: Click chemistry crosslinking reactions using azido-dPEG®₁₁-amine. In this scheme, both copper-catalyzed and copper-free click chemistry reactions are shown.

The crosslinking properties of azido-dPEG®₁₁-amine give it utility in a wide range of applications, including surface modification, small molecule crosslinking, and atomic force microscopy.

Surface modification

Non-specific interactions are a common frustration for scientists. Proteins, peptides, and nucleic acids stick randomly to uncoated metal, plastic, or glass surfaces, and can lead to erroneous outcomes. This problem complicates western blots, ELISAs, the analysis of column fractions collected in plastic or glass tubes, and many other areas of study. Several methods exist to reduce or eliminate these non-specific interactions. These methods include coating glass surfaces with sugars ^[4], silanization ^[5], and many types of PEGylation ^[6] ^[7]. Suppose, though, that you want to eliminate non-specific interactions and, at the same time, coat your surface with a reactive coating that allows for further modification? Using the crosslinker, azido-dPEG^®₁₁-amine, you functionalize the surface with carboxylic acid groups, activate them, and then react the crosslinker with the activated surface. The result is shown in Figure 2.

Surface Functionalized with Carboxylic Acid Groups

Surface now PEGylated and azidoated for click chemistry reactions

Figure 2: Surface coating with azido-dPEG®₁₁-amine.

A dense coating of dPEG molecules reduces or eliminates non-specific binding. This coating, though, leaves many closely spaced azide groups sticking up from the surface. These azide groups can react with some target molecules (a small molecule, peptide, or protein into which an alkyne group has been installed). However, steric hindrance (crowding) prevents most of the azide groups from reacting with the target alkyne. The unreacted azides are effectively wasted.

Figure 3: Product QBD-10278 m-dPEG®₈-amine.

A better way to coat the surface is to mix the crosslinker with a methoxy-terminated dPEG amine. In this scenario, combining m-dPEG^®₈-amine (QBD-10278) (29.7 Å )(Figure 3) in a ratio of >3:1 with azido-dPEG®₁₁-amine (44.2 Å ) crosslinker will achieve the results shown in Figure 4.

Surface Functionalized with Carboxylic Acid Groups

Surface now PEGylated, partly azidoated for click chemistryreactions, and fully blocked with m-DPEG®₈ to prevent non-specific interactions with the surface.

Figure 4: Mixed surface coating of azido-dPEG®₁₁-amine and m-dPEG®₈-amine.

This type of coating reduces non-specific binding. Fewer azide groups on the surface result in less crowding of the subsequent alkyne reactants. In turn, reduced steric crowding facilitates the introduction of large molecules, such as peptides and proteins, onto the surface. Thus, the decreased steric hindrance may prove particularly advantageous when carrying out SPAAC click chemistry with cyclooctyne groups that are often bulky.

Protein/Peptide/Small Molecule Crosslinking

Dr. Ravi S. Kane, formerly at Rensselaer Polytechnic Institute, used structure-based design to develop a heptavalent anthrax toxin inhibitor ^[8]. The inhibitor consisted of a seven-membered β-cyclodextrin core. The primary hydroxyl groups of the β-cyclodextrin core were then converted to terminal alkyne groups by reaction with propargyl bromide. Using copper-catalyzed click chemistry, Dr. Kane’s group reacted the heptavalent alkyne with azido-dPEG^®₁₁-amine in high yield. Next, they reacted the free amines, first with chloroacetic anhydride and then with a peptide that modeling studies strongly suggested would inhibit the formation of anthrax toxin (Figure 5).

Figure 5: The reaction scheme used to develop a heptavalent anthrax toxin inhibitor: (a) TBDMSCI, pyridine, 0 ˚C -rt; (b) NaH, MeI, THF; (c) NH₄F, MeOH, reflux; (d) NaH, propargyl bromide, DMF, 0 ˚C -rt; (e) CuSO₄, sodium ascorbate, THF/H₂O/t-BuOH (0.5:1:1), 80 ˚C; (f) chloroacetic anhydride, triethylamine; and (g) peptide, DMF, DBU, triethylamine. This image is taken from reference ^[7], below, and is reprinted by permission, copyright 2011, American Chemical Society.

The result was a well-defined macromolecular structure with precise spatial control that appeared from modeling studies to be able to inhibit the formation of anthrax toxin (Figure 6).

Figure 6: A heptavalent anthrax toxin inhibitor built by structure-based design using BioDesign’s azido-dPEG®₁₁-amine: (a) Structure of the LF-binding face of [PA₆₃]₇. Residues 184, 187, 197, and 200, which from part of the peptide-binding site are shown in purple. (b) Structure of the core, β-cyclodextrin. (c) Scheme illustrating the binding to [PA₆₃]₇ of a heptavalent inhibitor, synthesized by the attachment of seven inhibitory peptides to the β-cyclodextrin core via an appropriate polyethylene glycol linker. This image is taken from reference 8, below, and is reprinted by permission, copyright 2011, American Chemical Society.

In testing the heptavalent anthrax toxin inhibitor, six out of seven rats treated with the inhibitor and exposed to anthrax did not develop anthrax. Conversely, rats exposed to anthrax and either not treated with inhibitor or treated with a placebo inhibitor developed anthrax symptoms and died.

CuAAC is a simple reaction that gives high yields with minimal byproducts. This research shows the power of using click chemistry with a dPEG reagent to exert spatial control over macromolecular product design. The precise spatial control needed to position the inhibiting peptide from the β-cyclodextrin core would not have been possible with a dispersed PEG. Only with a single molecular weight PEG (dPEG) could that kind of control been obtained. Having a single molecular weight PEG product allows greater control over product design and purity. As demonstrated in the example, compared to traditional, dispersed PEGs, a dPEG compound simplifies product analysis.

Atomic Force Microscopy

Atomic Force Microscopy (AFM) is a type of scanning probe microscopy with a resolution to fractions of a nanometer. One application of AFM measures the force between a probe and a sample. This use of AFM is known as force spectroscopy, which is when “single (bio-) molecular bonds are actively broken to assess their range and strength” ^[9].

In single-molecule AFM force spectroscopy (SMFS), compounds of interest are attached by linkers to the cantilevers used to measure the force. Each cantilever attaches a single molecule ^[9], hence the name.

PEG is the most commonly used linker for attaching biomolecules to cantilevers ^[11] ^[12] ^[13]. However, PEG polymers are dispersed and have varied chain lengths ^[12] ^[14], making traditional PEG linkers less than ideal for SMFS.

A 2012 Master’s Thesis by Jamie Maciaszek in the lab of Yuri L. Lyubchenko reported that linkers containing a single molecular weight and chain length were superior to traditional, dispersed PEG linkers ^[14]. Moreover, using azido-dPEG^®₁₁-amine SMFS research in the lab of George Lykotrafitis detected and quantitatively mapped individual calcium-activated small conductance (SK) potassium channels in living neurons. The researchers joined the amine end of the crosslinker to APTES-activated silicon nitride cantilevers. Click chemistry crosslinking then linked the molecules of interest with the resulting azide-coated surface. The dPEG linker was ideal for the research due to the absence of chain length heterogeneity ^[15].

Conclusion

BioDesign click chemistry crosslinkers are designed for versatility and can be utilized in an assortment of applications. dPEG products, including azido-dPEG®₁₁-amine crosslinker, are available in different sizes to better suit specific requirements. Learn more about BioDesign and additional crosslinker options here.

References:

Hartmuth C. Kolb, M. G. Finn, and K. Barry Sharpless. Click Chemistry: Diverse Chemical Function from a Few Good Reactions. Angew. Chem. Int. Ed. (2001), 40, 2004-2021. [Wiley]
Chao-Jun Li and Barry M. Trost. Green chemistry for chemical synthesis. Proc. Nat. Acad. Sci. (September 9, 2008), 105(36), 13197-13202. [PNAS]
Greg T. Hermanson. “Chemoselective Ligation; Bioorthogonal Reagents,” in Bioconjugate Techniques, 3rd edition. New York: Academic Press, 2013, page 771. We at Quanta BioDesign recommend Greg’s book to all of our customers. You can buy it from us. To get started, please click this link and then click “Add to Cart” to order the book.
Gangadhar Jogikalmath. Method for blocking non-specific protein binding on a functionalized surface. US 20080213910 A1, September 4, 2008. [Google Patents]
Nick R. Glass, Ricky Tjeung, Peggy Chan, Leslie Y. Yeo, and James R. Friend. Organosilane deposition for microfluidic applications. Biomicrofluidics (2011), 5(3), 036501–036501-7. [PubMed]
Jacob Piehler, Andreas Brecht, Ramūnas Valiokas, Bo Liedberg , and Günter Gauglitz. A high-density poly(ethylene glycol) polymer brush for immobilization on glass-type surfaces. Biosensors & Bioelectronics (2000), 15, 473–481. [Research Gate]
Hongwei Chen, Julie Yeh, Liya Wang, Xinying Wu, Zehong Cao, Y. Andrew Wang, Minming Zhang, Lily Yang, and Hui Mao. Reducing Non-Specific Binding and Uptake of Nanoparticles and Improving Cell Targeting with an Antifouling PEO-b-PγMPS Copolymer Coating.. Biomaterials (July 2010), 31(20): 5397–5407. [PubMed]
Amit Joshi, Sandesh Kate, Vincent Poon, Dhananjoy Mondal, Mohan B. Boggara, Arundhati Saraph, Jacob T. Martin, Ryan McAlpine, Ryan Day, Angel E. Garcia, Jeremy Mogridge, and Ravi S. Kane. Structure-Based Design of a Heptavalent Anthrax Toxin Inhibitor. Biomacromolecules (2011), 12(3), 791–796. [ACS Publications]
Bullerjahn, J., Sturm, S. & Kroy, K. Theory of rapid force spectroscopy. Nat Commun 5, 4463 (2014). [Nature Communications]
Keir C. Neuman and Attila Nagy. Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy. Nature Methods (June 2008), 5(6), 491-505. [PubMed]
Bo-Hyun Kim, Nicholas Y Palermo, Sándor Lovas, Tatiana Zaikova, John Keana, and Yuri Lyubchenko. Single molecule atomic force microscopy force spectroscopy study of Aß-40 interactions. Biochemistry (2011), 50(23), 5154–5162. [ACS Publications]
Timothy V. Ratto, Kevin C. Langry, Robert E. Rudd, Rodney L. Balhorn, Michael J. Allen, Michael W. McElfresh. Force Spectroscopy of the Double-Tethered Concanavalin-A Mannose Bond. Biophysical Journal (2004), 86(4), 2430-2437. [ScienceDirect]
Zenghan Tong, Andrey Mikheikin, Alexey Krasnoslobodtsev, Zhengjian Lv, Yuri L. Lyubchenko. Novel polymer linkers for single molecule AFM force spectroscopy. Methods (April 2013), 60(2), 161-168. [PubMed]
Jamie L. Maciaszek. Detection of SK2 Channels on Hippocampal Neurons. Master’s Thesis. University of Connecticut Graduate School, 2012. [UCONN]
Jamie L. Maciaszek, Heun Soh, Randall S. Walikonis, Anastasios V. Tzingounis, and George Lykotrafitis. Topography of Native SK Channels Revealed by Force Nanoscopy in Living Neurons. The Journal of Neuroscience (2012), 32(33), 11435-11440. [PubMed]

December 23, 2024

SEARCH THE BLOG

Quantum Dots

Vector Laboratories R&D

Click Chemistry Crosslinking with dPEG®

Vector Laboratories R&D

Maleimide Crosslinker Selection Guide

Vector Laboratories R&D

It Takes Two to Tango, Part 1: Bioconjugation

Gowtham SP

It Takes Two to Tango, Part 2: Applications of Bioconjugation

Gowtham SP

The post Click Chemistry Crosslinking with dPEG® appeared first on VectorLabs.

Maleimide Crosslinker Selection Guide

Vector Laboratories R&D — Mon, 21 Oct 2024 22:28:41 +0000

Maleimide Crosslinker Selection Guide

Posted in: Tips and Tricks

Maleimide Crosslinker Selection Guide

Bioconjugate therapeutics are among the most promising new biopharmaceutical modalities and are receiving increased interest from biopharmaceutical and research organizations, with antibody drug conjugates (ADCs) leading the way in terms of active development and progress to the clinic. Despite the immense potential of ADCs, the off-target toxicity associated with early ADCs delayed the class’s expansion. In first-generation ADCs, researchers had yet to explore how linker chemistry and bioconjugation strategy impact the controlled delivery of drug payloads. Now, it is well understood that careful linker design is essential for effective tumor targeting properties without affecting the non-tumorous entity. Recent advancements in linker technologies and conjugation approaches reignited interest in ADC biopharmaceuticals and have been driving development ever since.

Vector Laboratories’ BioDesign™ offers an extensive catalog of crosslinkers, including many with maleimide functional groups on one or more ends of linear and multi-armed discrete PEG (dPEG®) products. These crosslinkers enable the conjugation of thiols with amines or thiols with other thiols. The maleimide functional group reacts selectively with free thiols below pH 7.5. At pH 7.5 and above, the maleimide double bond reacts competitively with free amines (for example, lysine side chains) and free thiols.

Selecting the optimal maleimide crosslinker does not need to be an arduous task. In this guide, we will discuss amine-reactive, homofunctional, and other types of maleimide crosslinkers, and provide details to assist in the selection process.

Amine-Reactive Maleimide Crosslinkers

Acid-Functionalized Maleimide Crosslinker Products

At Vector Laboratories, we offer single molecular weight maleimide-functionalized dPEG^® acids with lengths of 2, 4, 6, 8, 12, and 24 EO units. The terminal propionic group can be functionalized to form a variety of active esters. Alternatively, direct coupling of the acid to an amine is possible using DCC or EDC (often sold as the hydrochloride salt). Leaving the acid end unmodified after conjugating the maleimide moiety alters the conjugate’s surface charge. Surface charge modification may beneficially affect some drug or payload delivery applications.

Explore our MAL-dPEG^®-acid crosslinkers:

PN10265, MAL-dPEG^®₂-acid

PN10338, MAL-dPEG^®₄-acid

PN10065, MAL-dPEG^®₆-acid

PN10275, MAL-dPEG^®₈-acid

PN10285, MAL-dPEG^®₁₂-acid

PN10315, MAL-dPEG^®₂₄-acid

Figure 1: General structure of Vector Lab’s MAL-dPEG^®_n-acid crosslinkers.

NHS ester-Functionalized Maleimide Crosslinker Products

N-Hydroxysuccinimide (NHS) esters are among the most common reactive acylating agents presently used in bioconjugation.^[1] NHS esters were initially developed as homobifunctional crosslinkers.^[2]^,[3] Today, they are among the most widely used amine-reactive crosslinking agents in bioconjugation.¹

BioDesign MAL-dPEG^®-NHS esters provide quality and consistent performance in research and manufacturing applications. We offer our products with linker lengths of 2, 4, 6, 8, 12, and 24 ethylene oxide units. Please see Figure 2 below.

Figure 2: General structure of MAL-dPEG^®_n-NHS ester crosslinkers.

MAL-dPEG^®-NHS ester products:

PN10266, MAL-dPEG^®₂-NHS ester

PN10214, MAL-dPEG^®₄-NHS ester

PN10064, MAL-dPEG^®₆-NHS ester

PN10274, MAL-dPEG^®₈-NHS ester

PN10284, MAL-dPEG^®₁₂-NHS ester

PN10314, MAL-dPEG^®₂₄-NHS ester

TFP ester-Functionalized Maleimide Crosslinker Products

Active esters based on 2,3,5,6-tetrafluorophenol (TFP; see Figure 3) are an increasingly popular alternative to NHS esters.

Figure 3: 2,3,5,6-Tetrafluorophenol is used to make MAL-dPEG^®_n-TFP ester crosslinkers.

TFP esters are more stable to hydrolysis in aqueous media than NHS esters, as discussed below in more detail. Shorter MAL-dPEG^®-TFP esters may have slightly reduced water solubility due to the hydrophobic TFP moiety on one end of the crosslinker. However, using a dry water-miscible solvent such as DMAC or acetonitrile can overcome any reduced water solubility issue.

Figure 4: General structure of MAL-dPEG^®_n-TFP ester maleimide crosslinker products.

Figure 4 shows the general structure of seven of our eight MAL-dPEG^®-TFP esters, while Figure 5 shows the specific structure of one unique product, MAL-dPEG^®₂₄-amido-dPEG^®₂₄-TFP ester. We designed PN11303 to provide a very long linker (158 atoms, 183.5 Å) between a thiol and an amine.

Figure 5: Structure of PN11303, MAL-dPEG^®₂₄-amido-dPEG^®₂₄-TFP ester

One possible application for PN11303 exists in MAL-dPEG^®₂₄-amido-dPEG^®₂₄-DSPE (Figure 6), which is PN11303 conjugated to 1,2-Distearoylphosphatidylethanolamine (DSPE) for use in lipids and micelles. The long discrete PEG linker allows for the conjugation of a payload such as a small molecule drug, targeting peptide, antibody, antibody fragment, or other protein. Moreover, the unconjugated maleimide moiety at the surface of a liposome may promote thiol-mediated cell-membrane trafficking.^[4]^,[5] Other applications are certainly possible, limited only by imagination and nature’s laws.

Figure 6: Structure of PN11093, MAL-dPEG^®₂₄-amido-dPEG^®₂₄-DSPE. This product exemplifies an application of PN11303, discussed above. Please see the text for details.

TFP-functionalized MAL-dPEG^® crosslinker products:

PN10549, MAL-dPEG^®₂-TFP ester

PN10551, MAL-dPEG^®₄-TFP ester

PN10556, MAL-dPEG^®₆-TFP ester

PN10552, MAL-dPEG^®₈-TFP ester

PN10553, MAL-dPEG^®₁₂-TFP ester

PN10554, MAL-dPEG^®₂₄-TFP ester

PN10555, MAL-dPEG^®₃₆-TFP ester

PN11303, MAL-dPEG^®₂₄-amido-dPEG^®₂₄-TFP ester

TFP ester, NHS ester, and Acid-functionalized: Selecting a Terminal Group

Selecting between a TFP-, NHS-, or acid-terminated MAL-dPEG^® crosslinker requires a few considerations. Some customers choose crosslinkers based on their familiarity with the chemistry. NHS esters are the most common and most popular amine-reactive groups for reactive acylating agents. Thus, the chemistry is quite well-known, and NHS esters are selected based primarily on that awareness. While familiarity is reassuring, we strongly encourage customers to consider other factors as well.

One important factor to consider is whether you want to use an NHS or TFP ester. For example, a MAL-dPEG^® acid should be used if the goal is to couple the MAL-dPEG^®-acid via 1-hydroxybenzotriazole or another amine-reactive agent. Also, a carbodiimide can be used to couple a MAL-dPEG^® acid directly to an amine without the need to form an active ester. If the direct coupling of the acid to an amine is desired, the acid-functionalized crosslinker should be used.

In 2017, Jian Wang et al. confirmed experimentally that TFP esters were superior to NHS esters for PEGylation.^[6] The half-lives of both NHS and TFP esters decrease as the pH increases. However, TFP esters consistently display longer half-lives than NHS esters across a wide range of pH values. In aqueous media, the optimum pH for conjugating TFP esters to amines is higher (>7.5) than NHS esters (7.0-7.2).

For reactions in water or aqueous buffers, pH is another consideration for the maleimide group. At or above pH 7.5, the maleimide group reacts competitively with free amines, such as lysine side chains.¹ However, the maleimide-thiol reaction proceeds optimally at pH 6.5 – 7.5,¹,^[7^] while the reaction between TFP esters and free amines is very slow below pH 7.0.^[8] In our experience, NHS esters should be used at pH 7.0 if the ester must be reacted before the maleimide. Conversely, if the maleimide can be reacted first, it may be possible to use a TFP ester provided that the maleimide-thiol reaction is conducted at pH < 7 to prevent unwanted TFP ester hydrolysis or acylation of amines by the TFP ester.

The water solubility of TFP esters should also be considered. Our shortest MAL-dPEG^®-TFP ester products are less soluble in water than the corresponding MAL-dPEG^®-NHS esters. The use of water-miscible organic solvents such as DMAC or acetonitrile may permit product dissolution, allowing the reaction to proceed; however, in cases where organic solvents are impermissible, NHS esters may be preferred over TFP esters. Water solubility is generally not an issue for TFP esters with dPEG^®₈ and longer linker chains.

The choice of solvent for the reaction is critical to success. In addition to choosing the correct pH for aqueous reactions, users should ensure that aqueous buffers contain neither free amines nor free thiols that can react with the crosslinkers. We recommend using dry solvents for reactions run entirely in organic solvents (drying for >48 hours over activated 3 Å molecular sieves is sufficient) free of amines and thiols. If an organic base is required to drive the reaction of the active ester with free amines, we recommend a highly hindered base such as 2,6-lutidine over more commonly used organic bases such as triethylamine.

4-formyl-benzamido-dPEG^®₁₂-EDA-MAL: a maleimide crosslinker that can form a Schiff base, secondary amine, hydrazone, or oxime bond

4-formyl-benzamido-dPEG^®₁₂-EDA-MAL, product number QBD-10075 (Figure 7), is another unique product in the BioDesign catalog. Like our MAL-dPEG^®-acid and MAL-dPEG^®-NHS/TFP ester products, PN10075 crosslinks amines and thiols. The bond formed between an aldehyde and an amine imine is known as a Schiff base. The imine reduces to a secondary amine with various reducing agents.

Figure 7: Full-length (top) and abbreviated (bottom) structure of PN10075, 4-formyl-benzamido-dPEG^®₁₂-EDA-MAL. This product reacts with amines to form imines (Schiff bases), hydrazides to form hydrazones, and aminooxy compounds to form oxime bonds. Please see the text for details.

Imine chemistry is dynamic, and that dynamism has proven highly valuable to synthetic chemists constructing supramolecular materials and stable exotic materials via dynamic covalent chemistry. A 2012 tutorial review by Matthew E. Belowich and Sir Fraser Stoddart provides excellent, clear details on dynamic imine chemistry.^[9]

If the aldehyde end of PN10075 reacts with a hydrazide, it forms a hydrazone, which is stable in water but reverts to starting materials in the presence of mild aqueous acid. Hydrazone bonds are useful for forming prodrugs and other constructs that should degrade in acidic environments. If PN10075 reacts with an oxyamine, the bond formed is an oxime, which is more stable than a hydrazone bond.

Figure 8 shows the reaction between an amine and PN10075. The imine-forming reaction proceeds most quickly at pH 3 – 5 but can proceed at higher pH values with the aid of a catalyst.^[10]^,[11],[12] High pH imine-forming reactions may necessitate reacting the maleimide end of PN10075 before the aldehyde end to avoid reactions between the maleimide and free amines. However, low pH reactions may not necessarily require that the maleimide-thiol conjugation occurs before the imine-forming reaction.

Figure 8: The reaction of PN10075, 4-formyl-benzamido-dPEG^®₁₂-MAL with an amine to form an imine (Schiff base).

Imines hydrolyze readily to starting materials in the presence of water. If aqueous stability is required, then the imine must be reduced to a secondary amine. Many agents reduce Schiff bases to secondary amines, including H₂, borane, borohydrides, and silanes.^[13]^,[14] Reductive amination of imines to secondary amines commonly uses sodium borohydride.^[15] However, sodium borohydride also reduces aldehydes and ketones that may be present in the molecule. Milder reducing agents such as sodium cyanoborohydride^[16]^,[17] or sodium triacetoxyborohydride^[18] may be required. Water and protic solvents rapidly destroy sodium triacetoxyborohydride,^[19] which may necessitate changes in the reaction setup.

Figure 9 shows an example reduction of an imine-containing conjugate of PN10075. In the example, the maleimide group is conjugated to a thiol.

Figure 9: Reduction of an imine (Schiff base) to a secondary amine with sodium cyanoborohydride.

Homofunctional Maleimide Crosslinker Products

Vector Laboratories offers a variety of BioDesign crosslinkers where all the terminal ends contain maleimide functional groups. We offer two homobifunctional maleimide products (two maleimide ends on a linear, discrete PEG), three homotetrafunctional maleimide products (four-armed discrete PEG), and one homohexafunctional maleimide product (six-armed discrete PEG). We build these products using our discrete PEG products. Two of the three homotetrafunctional maleimide products contain a pentaerythritol core, and one contains a tris core (Figure 10). The homohexafunctional maleimide product, PN11436, Hexa(-dPEG^®₁₁-MAL)dipentaerythritol, is built on a dipentaerythritol core (Figure 11).

Figure 10: Structures of Vector Laboratories’ homotetrafunctional maleimide crosslinker products. Pentaerythritol forms the core of the left structure, while the right structure uses a tris core.

Figure 11: Structure of Vector Laboratories’ crosslinker, PN11436, Hexa(-amido-dPEG^®₁₁-MAL)dipentaerythritol.

Homobifunctional Maleimide Crosslinkers

PN10215, Bis-MAL-dPEG^®₃

PN10397, Bis-MAL-dPEG^®₁₁

Homotetrafunctional Maleimide Crosslinkers

PN11433, MAL-dPEG^®₁₂-Tris(-dPEG^®₁₁-MAL)₃ (Tris core)

PN11435, Tetra(-amido-dPEG^®₁₁-MAL)pentaerythritol (pentaerythritol core)

PN11414, Tetra(-amido-dPEG^®₂₃-MAL)pentaerythritol (pentaerythritol core)

Other Maleimide Crosslinkers

Vector Laboratories offers two unique PEG-based maleimide crosslinkers built around a lysine core (Figure 12). bis-MAL-Lysine-dPEG^®₄-acid (Figure 12a), and bis-MAL-Lysine-dPEG^®₄-TFP ester (Figure 12b), both contain a dPEG^®₄ linker conjugated to the acid terminus of lysine and maleimidopropyl groups conjugated to each amine group of lysine. PN10630 has a propionic acid reactive terminus, while PN10631 is functionalized as a TFP ester.

Figure 12: Chemical structures of Bis-MAL-Lysine crosslinker products. a. PN10630, Bis-MAL-Lysine-dPEG^®₄-acid; b. PN10631, Bis-MAL-Lysine-dPEG^®₄-TFP ester.

The bis-maleimido groups can bridge disulfide bonds or, as shown by Tae Hyung Kim, Hai Hua Jiang, Seulki Lee, et al., conjugate two mono-sulfhydryl Exendin-4 peptides to one discrete PEG linker.^[20] Moreover, PN10630 forms an essential part of two of our PK/BD modifier products, Bis-MAL-Lysine-dPEG^®₄-dPEG^®₁₂-Tris(-dPEG^®₂₄-acid)₃, and Bis-MAL-Lysine-dPEG^®₄-dPEG^®₁₂-Tris(m-dPEG^®₂₄)₃.

Vector Laboratories also offers a few maleimide crosslinker products containing no discrete PEG linker. These compounds can be building blocks for PEGylated crosslinkers or standalone crosslinkers for specific applications.

Figure 13 shows one example of a non-PEGylated maleimide crosslinker, Bis-Maleimide Amine, TFA salt, product number QBD-10232. This product contains bis-maleimide-functionalized lysine, similar to the PEGylated crosslinkers discussed above. However, 1,6-diaminohexane replaces the dPEG^®₄ linker of PN10630 and PN10631 as the linker arm in this product. PN10232 is quite hydrophobic and offers none of the benefits of our maleimide crosslinker products functionalized with discrete PEG.

Figure 13: Structure of PN10232, Bis-Maleimide Amine, TFA salt, a non-PEGylated maleimide crosslinker product.

A 2012 study by Scheer, Sandoval, Elliott, et al. successfully used PN10232 to design trastuzumab variants with customized F(ab’)2 domains. In this study, the authors introduced single cysteine substitutions into the V_H and V_L regions of trastuzumab’s Fab region. PN10232 then crosslinked different Fab units, creating custom-made F(ab’)2 units with new functions.^[21]

Figure 14: MPS-acid, product number 10323, also known as 3-maleimidopropionic acid.

For customers who want to build their own maleimide-functionalized products or use maleimide alone as a very short crosslinker, Vector Laboratories offers 3-maleimidopropionic acid, product number QBD-10323, which we call MPS-acid (Figure 14), and NHS-3-maleimidopropionate, product number 10217, which we call MPS (Figure 15).

Figure 15: PN10217, MPS, also known as NHS-3-maleimidopropionate.

Product numbers QBD-10323 and QBD-10217 both react with thiol at the maleimide olefin bond. With PN10323, functionalizing the terminal acid with an alkylating agent such as NHS, TFP, or HOBt permits cross-coupling to an amine. However, using a carbodiimide such as EDC permits direct coupling of the terminal acid to a free amine. PN10217 can be directly reacted with an amine using a hindered base such as 2,6-lutidine, to drive the reaction. PN10323 and PN10217 are useful as very short crosslinkers and building blocks for new, longer crosslinkers.

Figure 16: Product number 10177, MPS-EDA∙TFA.

Finally, we offer product number QBD-10177, MPS-EDA∙TFA (Figure 16), a short, non-PEGylated maleimide crosslinker product. This product crosslinks a sulfhydryl group with a carboxylic acid, or it can be used as a building block with discrete PEG to form a new crosslinker having a longer spacer between the maleimide group and the PEG linker.

Other Thiol Reactive Maleimide Crosslinker Products

PN10630, bis-MAL-Lysine-dPEG^®₄-acid

PN10631, bis-MAL-Lysine-dPEG^®₄-TFP ester

PN10232, Bis-Maleimide Amine, TFA salt

PN10323, MPS-acid

PN10217, MPS

PN10177, MPS-EDA∙TFA

Conclusion

BioDesign, by Vector Laboratories, offers an extensive catalog of maleimide crosslinkers that enable the conjugation of thiols with amines or thiols with other thiols. Numerous product applications exist for these crosslinkers, including antibody-drug conjugates (ADCs), fragment-drug conjugates (FDCs), protein-complex proximity analysis, FRET applications, modification of thiol-functionalized surfaces, supramolecular materials construction, and many more.

The maleimide crosslinkers available in the BioDesign portfolio of products benefit from our dPEG^® technology. Discrete PEG products improve the hydrophilicity of poorly water-soluble products and reduce the immunogenicity of potentially antigenic compounds. Because these products are single molecular weight products, the analysis of bioconjugates is simplified compared to traditional, non-uniform (i.e., polydispersed) PEG products.

For more than two decades, Vector Laboratories’ maleimide-dPEG^® crosslinkers have been trusted by researchers and industry professionals for use in diagnostic and therapeutic applications. We invite you to consider our highly cited BioDesign products and discover the dPEG^® difference!

References

[1] Hermanson, G. T. Chapter 3, The Reactions of Bioconjugation. In Bioconjugate Techniques; Academic Press: New York, NY, 2013; pp 229-258. Greg’s book is the industry-standard reference in the science and technology of bioconjugation, and we sell it on our website.

[2] Bragg, P. D.; Hou, C. Subunit Composition, Function, and Spatial Arrangement in the Ca²⁺– and Mg²⁺-Activated Adenosine Triphosphatases of Escherichia Coli and Salmonella Typhimurium. Archives of Biochemistry and Biophysics 1975, 167(1), 311-321. https://doi.org/10.1016/0003-9861(75)90467-1.

[3] Lomant, A. J.; Fairbanks, G. Chemical Probes of Extended Biological Structures: Synthesis and Properties of the Cleavable Protein Cross-Linking Reagent [³⁵S]Dithiobis(Succinimidyl Propionate). Journal of Molecular Biology 1976, 104(1), 243-261. https://doi.org/10.1016/0022-2836(76)90011-5.

[4] Li, T.; Takeoka, S. Enhanced Cellular Uptake of Maleimide-Modified Liposomes via Thiol-Mediated Transport. Int. J. Nanomed. 2014, 9(1), 2849-2861. https://doi.org/10.2147/IJN.S58540

[5] Che, J.; Okeke, C. I.; Hu, Z.-B.; Xu, J. DSPE-PEG: A Distinctive Component in Drug Delivery System. Curr. Pharm. Des. 2015, 21(12), 1598-1605.

[6] Wang, J.; Zhang, R.-Y.; Wang, Y.-C.; Chen, X.-Z.; Yin, X.-G.; Du, J.-J.; Lei, Z.; Xin, L.-M.; Gao, X.-F.; Liu, Z.; Guo, J. Polyfluorophenyl Ester-Terminated Homobifunctional Cross-Linkers for Protein Conjugation. Synlett 2017, 28(15), 1934-1938. https://doi.org/10.1055/s-0036-1590974.

[7] Partis, M. D.; Griffiths, D. G.; Roberts, G. C.; Beechey, R. B. Cross-Linking of Protein by ω-Maleimido Alkanoyl N-Hydroxysuccinimido Esters. J Protein Chem 1983, 2(3), 263-277. https://doi.org/10.1007/BF01025358.

[8] Quanta BioDesign, personal observations with TFP ester reactions, 2019.

[9] Belowich, M. E.; Stoddart, J. F. Dynamic Imine Chemistry. Chem. Soc. Rev. 2012, 41(6), 2003-2024. https://doi.org/10.1039/C2CS15305J.

[10] Wendeler, M.; Grinberg, L.; Wang, X.; Dawson, P. E.; Baca, M. Enhanced Catalysis of Oxime-Based Bioconjugations by Substituted Anilines. Bioconjugate Chem. 2014, 25(1), 93-101. https://doi.org/10.1021/bc400380f.

[11] Larsen, D.; Kietrys, A. M.; Clark, S. A.; Park, H. S.; Ekebergh, A.; Kool, E. T. Exceptionally Rapid Oxime and Hydrazone Formation Promoted by Catalytic Amine Buffers with Low Toxicity. Chem. Sci. 2018, 9(23), 5252-5259. https://doi.org/10.1039/C8SC01082J.

[12] Kölmel, D. K.; Kool, E. T. Oximes and Hydrazones in Bioconjugation: Mechanism and Catalysis. Chem. Rev. 2017, 117(15), 10358-10376. https://doi.org/10.1021/acs.chemrev.7b00090.

[13] Baxter, E. W.; Reitz, A. B. Reductive Aminations of Carbonyl Compounds with Borohydride and Borane Reducing Agents. In Organic Reactions; American Cancer Society, 2004; pp 1-714. https://doi.org/10.1002/0471264180.or059.01.

[14] Fondo, M.; Corredoira-Vázquez, J.; GarcíaDeibe, A. M.; Sanmartín-Matalobos, J. An Easy Approach to Obtain Alcohol-Amines by Reduction of Alcohol Functionalized Imines. Proceedings 2019, 9(1), 2. https://doi.org/10.3390/ecsoc-22-05697.

[15] Billman, J. H.; Diesing, A. C. Reduction of Schiff Bases with Sodium Borohydride. J. Org. Chem. 1957, 22(9), 1068-1070. https://doi.org/10.1021/jo01360a019.

[16] Hutchins, R. O.; Hutchins, M. K.; Crawley, M. L.; Mercado-Marin, E. V.; Sarpong, R. Sodium Cyanoborohydride. In Encyclopedia of Reagents for Organic Synthesis; American Cancer Society, 2016; pp 1-14. https://doi.org/10.1002/047084289X.rs059.pub3.

[17] Tadanier, J.; Hallas, R.; Martin, J. R.; Stanaszek, R. S. Observations Relevant to the Mechanism of the Reductive Aminations of Ketones with Sodium Cyanoborohydride and Ammonium Acetate. Tetrahedron 1981, 37(7), 1309-1316. https://doi.org/10.1016/S0040-4020(01)92446-9.

[18] Abdel-Magid, A. F.; Carson, K. G.; Harris, B. D.; Maryanoff, C. A.; Shah, R. D. Reductive Amination of Aldehydes and Ketones with Sodium Triacetoxyborohydride. Studies on Direct and Indirect Reductive Amination Procedures1. J. Org. Chem. 1996, 61 (11), 3849-3862. https://doi.org/10.1021/jo960057x.

[19] Gribble, G. W.; Abdel-Magid, A. F. Sodium Triacetoxyborohydride. In Encyclopedia of Reagents for Organic Synthesis; American Cancer Society, 2007. https://doi.org/10.1002/9780470842898.rs112.pub2.

[20] Kim, T. H.; Jiang, H. H.; Lee, S.; Youn, Y. S.; Park, C. W.; Byun, Y.; Chen, X.; Lee, K. C. Mono-PEGylated Dimeric Exendin-4 as High Receptor Binding and Long-Acting Conjugates for Type 2 Anti-Diabetes Therapeutics. Bioconjugate Chem. 2011, 22(4), 625-632. https://doi.org/10.1021/bc100404x.

[21] Scheer, J. M.; Sandoval, W.; Elliott, J. M.; Shao, L.; Luis, E.; Lewin-Koh, S.-C.; Schaefer, G.; Vandlen, R. Reorienting the Fab Domains of Trastuzumab Results in Potent HER2 Activators. PLOS ONE 2012, 7(12), e51817. https://doi.org/10.1371/journal.pone.0051817.

October 21, 2024

SEARCH THE BLOG

Quantum Dots

Vector Laboratories R&D

Click Chemistry Crosslinking with dPEG®

Vector Laboratories R&D

Maleimide Crosslinker Selection Guide

Vector Laboratories R&D

It Takes Two to Tango, Part 1: Bioconjugation

Gowtham SP

It Takes Two to Tango, Part 2: Applications of Bioconjugation

Gowtham SP

The post Maleimide Crosslinker Selection Guide appeared first on VectorLabs.

Breaking Barriers in STEM: Embracing Diversity and Cultivating Inclusive Spaces

Vector Laboratories R&D — Wed, 21 Jun 2023 19:50:37 +0000

Breaking Barriers in STEM: Embracing Diversity and Cultivating Inclusive Spaces

Melina Villaseñor, Production Supervisor at Vector Laboratories

My name is Melina Villasenor, and I am a production supervisor for the formulation of products at Vector Laboratories. New to the biotech and sciences professional space, most of my time is spent in or around lab spaces, yet labs tend not to have people that look like me.

As a Mexican-American, I stand among a limited number of Latinx folks striding forward into the realm of sciences. The good news is, our representation has seen a gradual rise over the years, thanks in large part to the increasing visibility of successful Latinx professionals in STEM fields. Nevertheless, on a typical day, it’s just me and three other Latinx colleagues amongst the many other people making regular appearances in the lab.

As a Latinx, queer, non-binary, disabled individual, it has always been rare for me to see people in STEM who “look like me.” It reminds me of a piece of wisdom shared by a mentor: “Life happens somewhere in the middle;” when it comes to diversity, this couldn’t be truer. We are built to exist in intersections, and recognizing the way humans are multi-faceted in experiences, expression, culture, etc., helps us create unique spaces full of growth opportunities. As humans, we all fall into different diversity categories, and so creating space for more people that “look like me” allows for a more legitimate, human experience in the workspace.

That said, it’s not as simple as it sounds. Ensuring representation in STEM fields requires equitable opportunities for underrepresented groups. It starts by nurturing young minds, encouraging them to dream freely, and shielding them from the discriminations that marginalized groups often face that prevent them from even starting the journey down a STEM path.

My high school chemistry teacher, Mr. Escobar, an immigrant from El Salvador, was a beacon of encouragement as I pursued my love for science amidst a sea of challenges. An encounter with my principal, who tried to dissuade me from taking AP Chemistry due to its perceived difficulty and the possibility of extra funding should the primarily minority class be dismantled from a single student dropping it, threatened to derail my enthusiasm. However, it was Mr. Escobar’s unwavering faith in his students’ potential, combined with his own success story in the STEM field, that truly bolstered my resolve to continue on this challenging yet rewarding path.

As I’ve progressed in my career, I’ve met many individuals from marginalized communities, each with a similar narrative. A mentor who believed in them, a professor who understood them, and numerous hurdles they overcame. It’s a shared sentiment among us that having representation from the get-go would have made our journeys less daunting. But we undertake these challenges with the resolve to become the representation we lacked, to inspire the next generation.

Today, we find ourselves guiding peers, supporting local communities, and mentoring the younger generation to traverse the path we once took to our careers in STEM. Providing mock interviews and resume reviews can be especially beneficial for college students struggling to make connections and advocate for themselves during job hunting. Outreach and mentorship programs can be even more impactful, offering young individuals a glimpse into their potential STEM career path, and equipping them with the tools, tips, and support they might not have had otherwise.

Addressing diversity requires an equitable approach. Simply treating everyone equally, by assuming everyone has had the same experiences or journey in a STEM career, can unintentionally undermine the unique impact of diversity on individuals and within both corporate and academic spaces. It’s equity, rather, that lets us extend an extra hand, allocate more resources, and provide additional funding to those groups who need it the most.

My hopes lie in the community marginalized groups can bring to each other and in companies supporting these efforts by proudly presenting as allies to minority groups and funneling funds into communities with high populations of minorities who might not have the necessary tools to thrive. Champion diversity in STEM spaces. Encourage marginalized voices to speak about their experiences. Include them in talent acquisition, in decision-making, and in more than just the celebration of cultural history months.

Actively advocating for diversity is key. We must resist falling into the trap of the bystander effect, simply assuming that someone else will instigate the needed changes. The responsibility and opportunity lie with each one of us, shaping our individual paths while collectively transforming the landscape of STEM for the generations to come.

June 21, 2023

SEARCH THE BLOG

Quantum Dots

Vector Laboratories R&D

Click Chemistry Crosslinking with dPEG®

Vector Laboratories R&D

Maleimide Crosslinker Selection Guide

Vector Laboratories R&D

It Takes Two to Tango, Part 1: Bioconjugation

Gowtham SP

It Takes Two to Tango, Part 2: Applications of Bioconjugation

Gowtham SP

The post Breaking Barriers in STEM: Embracing Diversity and Cultivating Inclusive Spaces appeared first on VectorLabs.

The Waiting Game: A Search for a Streamlined IHC Protocol

Vector Laboratories R&D — Wed, 11 Aug 2021 03:06:00 +0000

The Waiting Game: A Search for a Streamlined IHC Protocol

Picture this: a chemist walks into a lab and asks his colleagues how to transition some immunohistochemistry (IHC)-stained slides in a buffer solution to coverslip with a non-aqueous mounting media. From his perspective, it’s a simple problem, and it should have a similarly simple solution. His colleague’s response, however? The process requires a complicated series of dehydration and xylene steps, which will take over half an hour. As you might imagine, this answer set off a strong response with Timothy Karpishin, PhD, Director of Chemistry at Vector Laboratories.

Thus, the chemist, now faced with a challenge, set out to change the way researchers have been handling IHC slides for generations.

The mismatch challenge

The problem for researchers working with IHC-stained slides is that you typically mount, or apply the coverslip, with a non-aqueous, very hydrophobic mounting medium. “This generally means you have to take your tissues, which are in a water-based solution, and change their environment by bringing them through a dehydration series to slowly remove the water,” explains Tim. “And then the mounting medium at the end of the process isn’t compatible with the alcohol you’re using — you still have to remove the alcohol and replace it with a hydrophobic liquid, such as xylene. This hydrophobic liquid is then compatible with your mounting media. So, faced with this tedious workflow, I suggested that we explore developing a mounting medium that compresses the workflow dramatically.” And several rounds of formulation testing later, VectaMount^® Express Mounting Medium was born.

Streamline your workflow

Compared to the traditional workflow, researchers can skip the long dehydration and clearing steps, which can take anywhere from 15 to 30 minutes. With VectaMount Express, following staining and a water rinse to remove the salts from your buffer solution, you rapidly dehydrate your slides in isopropanol. “We prefer isopropanol because it dries a little bit slower,” explains Tim, “so when you finally lift your slides out of the isopropanol, it doesn’t dry as quickly as ethanol. This gives you more time and flexibility for applying your coverslip.”

The clearing step that normally requires a xylene wash actually occurs when you apply VectaMount Express to your tissue specimen. “Clearing generally refers to matching the refractive index, or RI, of your tissue specimen with the mounting medium and glass slide,” explains Tim. Your glass slide and coverslip have an RI of about 1.5, and your tissue has an RI of about 1.46–1.48. To get clear, bright images, you need your mounting medium to have a similar RI. However, since alcohols, such as ethanol and isopropanol, have an RI of about 1.37, you can still see your tissue section on the slide after washing. That’s when xylene comes in. It matches your tissue with an RI of 1.49, making your tissue sections appear perfectly clear. VectaMount Express, on the other hand, was engineered with an RI of 1.49, but it is also compatible with alcohol. “That’s the difference,” says Tim. “You can clear your tissue without the need to swap out the alcohol. And when it dries, you’re left with a mounting medium that matches the RI of your tissue, maximizing the clarity, resolution, and brightness of the images.”

We asked Tim if he has any tips for researchers considering VectaMount Express; his advice, just give it a go—follow the instructions in the protocol and see what you can do with the saved time. “It simplifies the traditional workflow. There’s really nothing too complicated—no extra equipment is needed. It’s just a matter of the isopropanol washes and then the direct mounting,” says Tim. So far, the team has looked at a variety of substrates and counterstained slides 18 months after mounting with VectaMount Express, and the color and sensitivity remain stable.

Explore how you can streamline your IHC workflow with faster clearing and mounting. Get started today.

August 11, 2021

SEARCH THE BLOG

Quantum Dots

Vector Laboratories R&D

Click Chemistry Crosslinking with dPEG®

Vector Laboratories R&D

Maleimide Crosslinker Selection Guide

Vector Laboratories R&D

It Takes Two to Tango, Part 1: Bioconjugation

Gowtham SP

It Takes Two to Tango, Part 2: Applications of Bioconjugation

Gowtham SP

The post The Waiting Game: A Search for a Streamlined IHC Protocol appeared first on VectorLabs.

Dial up the Contrast with Counterstaining

Vector Laboratories R&D — Wed, 07 Jul 2021 03:22:00 +0000

Dial up the Contrast with Counterstaining

When scientists detect targets of interest with immunohistochemistry (IHC), antibody binding and substrate detection alone often produce a perplexing picture where the target lacks context. They need something to fill in the blanks, and that’s where counterstaining comes in. Think of it as a tour guide. Counterstaining IHC samples after enzymatic detection spatially orients the target within its surroundings, which can help guide researchers as they analyze their results. Counterstains bind cellular components to color the neighboring cellular structures, giving researchers a look at the surrounding tissue architecture, as well as a better understanding of the morphological and spatial context of the target.

Common Counterstain Choices

The most commonly used counterstains target nuclei in simple procedures. Hematoxylin stains chromatin within nuclei blue, which contrasts with dark-hued peroxidase and alkaline phosphatase substrate precipitates. There are various formulations of hematoxylin, including Mayer’s, Gill’s, Harris, and Ehrlich’s, that differ in how the compound is oxidized and the choice of mordant that sets the dye on the specimen. H & E staining, a common technique in histology, combines hematoxylin in combination with eosin stain, which colors the extracellular matrix and cytoplasm pink.

Some detection reagents produce blue or purple precipitates, making a hematoxylin stain incompatible. In this case, researchers can use methyl green, a light green stain that complements a wide range of enzyme substrates. However, methyl green requires heating for proper uptake to a specimen. Another alternative to hematoxylin is nuclear fast red, a pink stain that contrasts with blue- or brown-hued detection substrates. Applying nuclear fast red requires just one simple step and takes mere minutes.

Key Considerations for Using Counterstains

Researchers should carefully choose their counterstain based on compatibility with the detection substrate. Counterstains that deposit too much color or too similar a color to the detection substrate can obscure the target signal or surrounding tissue, causing false positive or negative results.

To control the final appearance, researchers use most counterstains in either a progressive manner, in which the stain incubation time controls the intensity of color, or a regressive manner, in which the user overstains the specimen and carefully removes excess stain with reagents, such as acetic acid, to achieve the desired color. The stain application method—dropping stain onto the slide or immersing slides fully into the stain in batches—can also affect the intensity and distribution of the counterstain. Batch staining ensures consistency if multiple slides are being stained at once.

Many aspects of the IHC protocol affect the final appearance of the specimen. For example, pH changes alter the color of hematoxylin, and alcohol solvents may affect the solubility or intensity of the primary stain. Also, the heat and acetone rinse steps of the methyl green staining protocol may dissolve or discolor certain substrates. Early in the IHC workflow, the thoroughness of the fixation step affects how well the specimen picks up the primary antibody and counterstain. Additionally, stains have trouble penetrating thick sections, but detergents such as Tween 20 can help with uptake. Finally, stains behave differently depending on the tissue or species type.

To navigate these challenges and ensure the best results, researchers should run controls on fresh tissue sections with fresh stain to see how well their chosen counterstain works. Ideally, researchers will achieve a light and even counterstain before proceeding with IHC to identify the antigen targets.

To Counterstain, or Not to Counterstain?

Some researchers add counterstains to their IHC slides simply out of habit. However, counterstaining is unnecessary in some cases. Experiments using single antibodies typically benefit from a counterstain, but if there are multiple antibody targets on one slide, adding an additional color may detract from both the signals of interest and the results. Researchers should ask themselves if they will gain additional information from a counterstain, or if the extra color will obscure their data.

Counterstains are very useful for accentuating the primary signal and contributing additional information about the target. However, researchers should carefully consider whether they need a counterstain and which formula is best for their sample. Taking the time to choose the proper counterstain can make for a much more effective IHC stain.

Explore more counterstaining tips and caveats in IHC in this on-demand webinar.

July 7, 2021

SEARCH THE BLOG

Quantum Dots

Vector Laboratories R&D

Click Chemistry Crosslinking with dPEG®

Vector Laboratories R&D

Maleimide Crosslinker Selection Guide

Vector Laboratories R&D

It Takes Two to Tango, Part 1: Bioconjugation

Gowtham SP

It Takes Two to Tango, Part 2: Applications of Bioconjugation

Gowtham SP

The post Dial up the Contrast with Counterstaining appeared first on VectorLabs.

Getting to the Specifics: Blocking for Immunohistochemistry

Vector Laboratories R&D — Wed, 23 Jun 2021 03:29:00 +0000

Getting to the Specifics: Blocking for Immunohistochemistry

Under perfect circumstances, chatting with a friend is clear and easy. But in a complex environment like a loud party, direct communication is often interrupted by competing sounds that drown out and muddle your speech. To get your message across, you need to find a way to cut through the background noise. Scientists performing antibody-based assays experience a similar challenge when analyzing their results at the end of a complicated staining protocol. To determine if what they are seeing is a real result, they need to block out the extraneous noise and focus on their specific signal.

Although immunohistochemistry (IHC) is a common technique, researchers often struggle with it due to the complexity of biological specimens. When performing IHC, scientists aspire to reduce background or non-specific staining on the specimen. The unwanted signal may be an overall weak stain across the tissue section or more intense coloration that appears specific and produces false positive results.

To gain a clear view, antibodies should bind their targets specifically, and the detection substrate should only precipitate at the site of antibody-bound antigens. However, non-specific binding of antibodies to tissue elements, such as charged particles, macromolecules, and Fc receptors, as well as endogenous enzyme activity often obscure specific antibody–antigen interactions. Blocking the sources of non-specific signals increases the signal-to-noise ratio for optimal results.

Protein Blockers

One major source of non-specific signal comes from intermolecular forces that promote antibody binding to other molecules in the specimen. To circumvent this, researchers add protein-based blocking agents that physically occupy non-specific binding sites prior to primary antibody addition. Depending on the blocking solution, the proteins within prevent non-specific binding to hydrophobic protein side chains or Fc receptors in the tissue. In theory, any protein that does not specifically bind to the epitope of interest can block unwanted interactions.

One common protein block is normal sera collected from healthy animals. Sera contain antibodies that bind non-specifically to Fc receptors, blocking the primary antibodies from interacting. The sera should originate from the species in which the secondary antibody was raised rather than that of the primary antibody animal. This ensures that the secondary antibody does not bind to the block and increase background signal.

Researchers also use protein block solutions, such as bovine serum albumin (BSA) and non-fat dry milk. BSA and non-fat dry milk proteins physically occupy binding spots that tend to interact non-specifically with antibodies through electrostatic and ionic interactions. An antibody specific for a target antigen should not have a greater affinity for the non-specific binding sites than the protein blockers. One advantage of this method is that there is no need to consider the source of the proteins in relation to the primary or secondary antibodies.

Animal-Free Blocker^® is a universal blocking agent and diluent perfect for researchers trying to avoid animal-derived proteins. An alternative to sera, BSA, casein, and non-fat dry milk, this reagent can be used for many applications, including IHC, nucleic acid blotting, and protein blotting.

Enzyme Blockers

Chromogenic detection methods use an enzyme linked to a secondary antibody to bind primary antibodies bound to the target. The most popular IHC detection substrates interact with horseradish peroxidase (HRP)– and alkaline phosphatase (AP)–conjugated antibodies. However, these enzymes are already present within some tissues, where they react with the detection substrate and cause background signal. To quench enzyme activity, enzyme blocking agents must be added prior to the detection step. Researchers often perform this blocking step before primary antibody incubation.

Peroxidase is especially common in the kidney, liver, and tissues containing red blood cells. Researchers often quench endogenous peroxidase with hydrogen peroxide. By simply dropping the chemical diluted in water, phosphate-buffered saline, or methanol onto prepared slides, the resulting reaction inactivates the enzyme and converts hydrogen peroxide to oxygen and water. Aqueous hydrogen peroxide solutions may destroy the underlying tissue architecture if the tissue is peroxidase-rich. In this case, methanol would be the better solvent choice.

If the detection substrate reacts with alkaline phosphatase, researchers may block endogenous enzyme activity with levamisole solution. Rather than applying this block prior to primary antibody addition, levamisole solution should be added to the alkaline phosphatase substrate solution right before detection.

BLOXALL^® Endogenous Blocking Solution is more effective than other enzyme blocking methods. It inactivates both endogenous peroxidase and alkaline phosphatase through a brief 10-minute incubation step. Many researchers prefer BLOXALL because it comes ready-to-use. Additionally, it is compatible with formalin-fixed, paraffin-embedded tissue sections, frozen tissue sections, and cell preparations.

Biotin-based detection systems in combination with peroxidase or alkaline phosphatase improve the sensitivity of IHC. Avidin- or streptavidin-biotinylated enzyme complexes tightly bind biotinylated antibodies. However, biotin is present in many tissues and can generate a background signal. Blocking with unconjugated avidin/streptavidin and then biotin works to block endogenous biotin, biotin receptors, and avidin binding sites.

Choosing the Right Strategy

No single protein block strategy is effective for all IHC experiments, so running controls is always important. Researchers must consider their antibodies, tissue type, and detection reagents to choose the best blocks for their experiments. Sections from the same specimen may exhibit dissimilar behavior due to differences in tissue or cell type, which display different binding sites and enzymes.

To troubleshoot and identify the source of background, scientists should first omit the primary antibody and see if they have a non-specific signal. If so, they can adjust wash times, check for species cross-reactivity, or try different protein blockers and buffers. Through this series of troubleshooting steps, researchers can identify the conditions that produce the best IHC results.

For more helpful tips and tricks be sure to check out our IHC Resource Guide.

Got more questions? Check out our FAQs or connect with an expert to learn more.

June 23, 2021

SEARCH THE BLOG

Quantum Dots

Vector Laboratories R&D

Click Chemistry Crosslinking with dPEG®

Vector Laboratories R&D

Maleimide Crosslinker Selection Guide

Vector Laboratories R&D

It Takes Two to Tango, Part 1: Bioconjugation

Gowtham SP

It Takes Two to Tango, Part 2: Applications of Bioconjugation

Gowtham SP

The post Getting to the Specifics: Blocking for Immunohistochemistry appeared first on VectorLabs.

An Introduction to Bioconjugation: Answering Your Questions

Vector Laboratories R&D — Wed, 09 Jun 2021 03:39:00 +0000

An Introduction to Bioconjugation: Answering Your Questions

Bioconjugation offers a way to chemically link two molecules to form a single hybrid, where at least one of the molecules in the partnership is a biomolecule. The resulting product retains the activity of each component, yet also gains a novel function that is not possible with either molecule alone. In a recent webinar, Dr. Craig Pow, Director, Technical Services at Vector Laboratories, provided an introduction to bioconjugation and its applications, as well as key factors to success. You can watch the entire on-demand webinar or keep reading for the answer to your questions.

If I am using expensive materials, oligonucleotides or antibodies, how do I maximize conjugate yield with the HyNic and 4FB chemistry?

What you want is to use a highly purified material—for oligos, pass it through high-performance liquid chromatography (HPLC), and you may want to desalt it as well.

To get rid of amine contaminants and preservatives, you may want to do a buffer exchange to prepare the buffer for subsequent bioconjugation. Vector Laboratories bioconjugation kits include Zeba™ desalting columns to enable that buffer exchange.

You may also want to make sure that your biomolecules are within the appropriate concentration range — use too little and you might not get appropriate labeling, use too much and you might have issues with solubility. Another consideration would be volume. Don’t use very small amounts because you might lose the material when you go through a purification step at the end when you remove unincorporated label. The opposite scenario, using too much material, might be problematic as well as it will reduce labeling efficiency. With HyNic and 4FB linkages, there are checkpoints during the bioconjugation methodology where you check the molar substitution ratio (MSR). These checkpoints enable you to assess how much HyNic and 4FB is attached to your molecules to ensure you have an appropriate MSR before you move forward with your bioconjugation reaction. You can then use the hydrazone bond or chromophore to both confirm the conjugation has occurred and quantify the amount. If you’re working with precious materials, this will give you the confidence to proceed.

What is the difference between the protein-oligo conjugation kit and the antibody-oligo kit?

These are different kits designed for each of these applications. The antibody kits have highly stringent steps in terms of purification, and include a purification step to clean up the labeled antibody. Antibodies are typically around 150 to 160 kD in size, therefore, an affinity resin is included in the antibody:oligo kit that binds to all mammalian IgG isotypes, ensuring that the conjugate is highly purified. With protein, there is often more variability in size, and one purification step may not fit all. Instead, your purification process will depend on the molecular weight of the protein you’re working with. I’d recommend looking at the protocols for each of these kits to better understand the parameters for consideration.

Why should I choose SoluLINK^® over other bioconjugation kits?

There are other methodologies for bioconjugation that were touched on during the webinar. SoluLINK chemistry, based on HyNic and 4FB, includes checkpoints, such as molar substitution ratio, as well as a hydrazone chromophore that allows you to do a quick UV scan to quantify that your conjugation was successful. This is invaluable for investigators who want consistency, reliability, and reproducibility. Some other kits on the market may have a shorter protocol, but there’s no cleanup step, so you can be left with residual material. If you’re working with particularly precious material or you need highly reproducible results, this purification step can be critical. Without it, you can run into problems with background, inconsistency, and poor labeling efficiency.

I’m interested in small interfering RNA (siRNA) antibody conjugates. Can you elaborate on this application and how you can remove excess siRNA?

Some investigators working with siRNA are trying to increase its longevity once injected. It can be cleared pretty quickly by either the kidneys or liver. The idea is to apply conjugation to a given target protein, so it can then be incorporated into target cells where it can perform its role of breaking down in a complementary RNA. Some researchers are looking at things called protein cages that actively protect siRNA and allow substantial amounts to get incorporated into the cell at the place of entry. For this application, you would need to follow the same methodologies as per the protein-oligonucleotide kit in terms of cleanup.

Watch the entire on-demand webinar for more information on bioconjugation, including case studies demonstrating how bioconjugation is being used to push the frontiers of both medicine and science, as well as an overview of the SoluLINK chemistry and Bioconjugation portfolio from Vector Laboratories. And be sure to check out our Bioconjugation Resource Guide to help you get started.

Got more questions? Check out our FAQs or connect with an expert to learn more.

June 9, 2021

SEARCH THE BLOG

Quantum Dots

Vector Laboratories R&D

Click Chemistry Crosslinking with dPEG®

Vector Laboratories R&D

Maleimide Crosslinker Selection Guide

Vector Laboratories R&D

It Takes Two to Tango, Part 1: Bioconjugation

Gowtham SP

It Takes Two to Tango, Part 2: Applications of Bioconjugation

Gowtham SP

The post An Introduction to Bioconjugation: Answering Your Questions appeared first on VectorLabs.

Improving IHC & IF Staining Results

Vector Laboratories R&D — Thu, 06 May 2021 03:54:00 +0000

Improving IHC & IF Staining Results

Immunohistochemistry (IHC) and immunofluorescence (IF) are widely used across the life sciences—in cancer research, immunology, neuroscience, cell biology, and histopathology—to help elucidate protein expression in cell and tissue specimens. And while these are well established and widely adopted techniques, when working with biological specimens, you can encounter common problems, such as background interference, lack of specificity, weak signal, or poor reproducibility.

In a recent webinar with The Scientist, Dr. Craig Pow, presented helpful tips and tricks for improving IHC and IF staining results. You can watch the full on-demand webinar or keep reading for some tips and tricks from the Q&A session.

How do the sensitivities of IHC and IF compare?

While the workflows for IHC and IF do have several similarities, it is can be a little bit like comparing apples and oranges. It is exceedingly difficult to draw a straight-line comparison between the two different applications.

Immunohistochemistry can be executed with a one- or two-step methodology followed by enzyme substrate detection with signal amplification, all while providing greater sensitivity with the two-step method.

Conventional immunofluorescence on the other hand, provides exceptionally fine, exquisite detail, depending on what you need to examine and hope to resolve.

For example, when studying subcellular localization, with multiplexing and immunofluorescence, you can easily colocalize expression of antigens in the same cell compartment of the same cell. You also can choose to implement amplified immunofluorescence approaches to increase sensitivity, for instance by opting to use VectaFluor™ Excel antibodies that can increase sensitivity by as much as three- to four-fold over fluorophore-conjugated secondary antibodies.

When it comes to detection, why should someone choose an alkaline phosphatase IHC detection method over one with horseradish peroxidase?

The use of peroxidase-based methodology, alongside a substrate such as 3,3′-diaminobenzidine (DAB), is probably the most used approach. It deposits a nice discrete colored precipitate for single protein detection. However, if you are working with tissue that contains endogenous peroxidase activity, such as highly vascularized material, you may need to add a quenching step. Quenching of the endogenous peroxidase can be managed by incubation with hydrogen peroxide solutions (3% H2O2 in water, 0.3% H2O2 in methanol) or commercial convenience products, such as BLOXALL^® Endogenous Blocking Solution, Peroxidase and Alkaline Phosphatase. While quenching can be effective, it does add more steps to the staining process.

For some tissues, switching to an alkaline phosphatase approach can circumvent the issues of persistent peroxidase activity. As an added benefit, the diffuse and translucent color of alkaline phosphatase substrates can better provide a clear view of the underlying tissue morphology. Alkaline phosphatase (AP) detection also provides a choice of several colors. For example, if you’re working with tissue with inherent pigmentation or melanoma, additional substrate color options for alkaline phosphatase detection enable broader spectrum staining in magenta, blue, indigo, and black. You can leverage these colorful options with double or triple staining. Explore protocols for chromogenic multiplexing in our IHC Multiplexing Guide.

How can I manage autofluorescence in my IF tissue preparation?

Autofluorescence or endogenous fluorescence may be inherent to your tissue or preparation method. It may arise from different cell types or multiple factors. For example, collagen, elastin, and red blood cells are particularly notorious for autofluorescence. Aldehyde-based fixatives, such as formalin, paraformaldehyde, or glutaraldehyde can induce autofluorescence. TrueVIEW® Autofluorescence reagents help diminish unwanted autofluorescence and dramatically improve signal-to-noise ratio.

TrueVIEW reagent is typically used after the secondary antibody incubation and subsequent washes. The reagent is applied for about two to five minutes, and then the sections are washed with PBS prior to mounting with anti-fade media.

Is it better to wash for a) fewer times for longer periods of time or b) more frequently for shorter periods of time?

While there’s no definitive answer for this, you will need to choose whatever is effective to help reduce toning, background, and remove the unbound material from your tissue preparation. At Vector Laboratories, we usually run two to three washes (2-5 minutes each) in between incubations. If you use thicker tissue sections, longer washes are required. Three to five washes (10–20 minutes each) are not unheard of for thicker sections, embryos, Xenopus, or zebrafish. Your best approach will start with these recommendations and optimize the steps to see what works best for you.

What other negative controls would be appropriate, beyond substituting nonimmune IgG?

Probably the best negative control for most would be to apply an irrelevant antibody, meaning something that will not bind to anything in your tissue preparation. Let’s say that you work with NeuN or GFAP on CNS or brain tissue. You know there won’t be any stratified keratinized epithelium in your tissue prep. In this scenario, you could select a primary antibody against stratified keratinized epithelium, raised in the same species as the isotype, at the same concentration, and then use typical detection reagents after that. This negative control would show that the primary antibody isotype from that species is not being bound inadvertently by some tissue or contributing to the staining.

Other negative control options may be used, such as if you’re researching a knockout model and you can test your antibody on that tissue. Or pre-immune serum, which is serum extracted from the animal prior to antibody immunization, could be applied instead of the primary antibody as a validation control to check for non-specific binding. Another control would be to incubate the primary antibody with the immunogen used to generate the antibody, but this approach may not be practical for those who use commercial antibodies.

Watch our on-demand Technique Talk webinar for more tips on identifying and eliminating sources of non-specific staining, as well as the appropriate controls to use. And be sure to check out our IHC Resource Guide and IF Resource Guide to help you navigate the workflows and select reagents.

Got more questions? Check out our FAQs or connect with an expert to learn more.

May 6, 2021

Jefferson Duarte – VectorLabs

Quantum Dots

Utilizing dPEG® Products in Quantum Dots Applications

What are Quantum Dots?

What is dPEG?

Quantum Dot Synthesis

Reducing Quantum Dot Non-Specific Binding with dPEG

Conclusion

Products

References:

CATEGORIES

SEARCH THE BLOG

TAGS

RECENT POSTS

Click Chemistry Crosslinking with dPEG®

Click Chemistry Crosslinking with dPEG®

What is dPEG?

What is click chemistry?

Why use click chemistry?

Click chemistry crosslinking ideas using dPEG reagents.

Surface modification

Protein/Peptide/Small Molecule Crosslinking

Atomic Force Microscopy

Conclusion

References:

CATEGORIES

SEARCH THE BLOG

TAGS

RECENT POSTS

Maleimide Crosslinker Selection Guide

Maleimide Crosslinker Selection Guide

Amine-Reactive Maleimide Crosslinkers

Acid-Functionalized Maleimide Crosslinker Products

NHS ester-Functionalized Maleimide Crosslinker Products

TFP ester-Functionalized Maleimide Crosslinker Products

TFP ester, NHS ester, and Acid-functionalized: Selecting a Terminal Group

Homofunctional Maleimide Crosslinker Products

Other Maleimide Crosslinkers

Conclusion

CATEGORIES

SEARCH THE BLOG

TAGS

RECENT POSTS

Breaking Barriers in STEM: Embracing Diversity and Cultivating Inclusive Spaces

Breaking Barriers in STEM: Embracing Diversity and Cultivating Inclusive Spaces

CATEGORIES

SEARCH THE BLOG

TAGS

RECENT POSTS

The Waiting Game: A Search for a Streamlined IHC Protocol

The Waiting Game: A Search for a Streamlined IHC Protocol

The mismatch challenge

Streamline your workflow

CATEGORIES

SEARCH THE BLOG

TAGS

RECENT POSTS

Dial up the Contrast with Counterstaining

Dial up the Contrast with Counterstaining

Common Counterstain Choices

Key Considerations for Using Counterstains

To Counterstain, or Not to Counterstain?

CATEGORIES

SEARCH THE BLOG

TAGS

RECENT POSTS

Getting to the Specifics: Blocking for Immunohistochemistry

Getting to the Specifics: Blocking for Immunohistochemistry

Protein Blockers

Enzyme Blockers

Choosing the Right Strategy

Got more questions? Check out our FAQs or connect with an expert to learn more.

CATEGORIES

SEARCH THE BLOG

TAGS

RECENT POSTS

An Introduction to Bioconjugation: Answering Your Questions

An Introduction to Bioconjugation: Answering Your Questions

If I am using expensive materials, oligonucleotides or antibodies, how do I maximize conjugate yield with the HyNic and 4FB chemistry?

What is the difference between the protein-oligo conjugation kit and the antibody-oligo kit?

Why should I choose SoluLINK^® over other bioconjugation kits?