In Situ Proximity Ligation – VectorLabs

ChromaLINK® Digoxigenin One-Shot™ Antibody Labeling Kit

Vector Laboratories R&D — Sat, 06 May 2023 02:33:47 +0000

Description

The ChromaLINK Digoxigenin One-Shot Antibody Labeling Kit provides convenient, consistent, and measurable digoxigenin labeling of 100 µg of antibody. Each kit contains ChromaLINK Digoxigenin, which incorporates a novel UV-traceable chromophore in the linker arm to enable reproducibility in your antibody labeling process. With a simple, non-destructive UV scan you can now quantify digoxigenin incorporation to ensure reproducible incorporation of the optimal number of haptens per antibody. Each ChromaLINK One-Shot kit contains everything needed to to label your antibody: buffers, reagents, desalting columns, and an easy-to-follow protocol and online DIG incorporation calculator.

Specifications


Conjugation Targets	Antibody
Label/Modifier Type	Digoxigenin
Reactivity	Amine
Recommended Storage	2° – 8°C – Do Not Freeze
Applications	In situ hybridization, Antibody Labeling, In Situ Proximity Ligation

Kit Components

Sulfo ChromaLINK® Digoxigenin (11 μg)
1X Modification Buffer (1.5 mL)
1X PBS (1.5 mL)
Anhydrous DMF (1.5 mL)
2 mL Collection Tube (4)
0.5 mL Zeba™ Desalting Column (2)
DIG-labeled Bovine IgG Control (100 μg)
1M Tris HCl (1.5 mL)

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Technical Information

Product Description

The ChromaLINK Digoxigenin One-Shot antibody labeling kit is specifically designed to incorporate a digoxigenin label into a single 100 microgram quantity of antibody resuspended in 100 μl of buffer. This kit comes complete with all the necessary components to label and rapidly quantify incorporated digoxigenin from a small quantity of antibody in about 90 minutes.

Digoxigenin has long been used as an alternative hapten reporter system to the streptavidin/biotin system (1). This system is based on a steroid isolated from the blossoms and leaves of the plant Digitalis purpurea, the only known natural source of digoxigenin. High affinity antibodies raised against digoxigenin thus exhibit virtually no cross-reaction with other biological molecules found in higher organisms. Both the specificity and sensitivity of this “parallel” hapten-reporter system often gives digoxigenin a major advantage over biotin-based detection systems. In more recent times, this reporter system has also been used in conjunction with the biotin-streptavidin system to detect multiplexed antigens (2). Unlike the biotin/streptavidin system, the digoxigenin label has never had a method for directly quantifying incorporation, until now.

The labeling process used in the ChromaLINK Digoxigenin One-Shot kit is based on a patented UV-traceable linker called Sulfo-ChromaLINK Digoxigenin (Figure 1). This advanced digoxigenin labeling reagent contains an aromatic water-soluble N-hydroxy-sulfosuccinimidyl ester functional group (a), which efficiently modifies protein lysine residues in a phosphate buffer system. The linker also possesses an embedded bis-aryl hydrazone structure (b), which forms the compound’s UV-traceable chromophore. This traceable signature enables the non-destructive and direct quantification of digoxigenin (c), attached to the antibody. These diverse chemical elements are linked together through a long-chain PEG3 spacer (d), which preserves antibody-digoxigenin binding affinity while simultaneously maintaining antibody solubility.
Figure 1: Structure of ChromaLINK Digoxigenin C52H67N6NaO17S; M.W. 1103.17

Benefits and Features

The ChromaLINK Digoxigenin One-Shot kit is a convenient, cost-effective way of incorporating digoxigenin into a small quantity (100 μg) of an antibody. After incorporation, digoxigenin is rapidly quantified using a non-destructive UV-scan (220-400 nm). The kit features high antibody recoveries (50-80%) with consistent digoxigenin incorporation. The degree of incorporation or molar substitution ratio (MSR) ranges from 2-8 digoxigenin molecules per antibody when used as directed.

The ChromaLINK Digoxigenin One-Shot kit can be used to label a diverse group of antibodies including all mammalian IgG, IgE, and avian IgY molecules. The ChromaLINK Digoxigenin One-Shot kit offers a robust method of both labeling and quantifying incorporated digoxigenin with a simple protocol. This “double-feature” provides researchers with greater confidence, consistency and reproducibility in all downstream applications.

Procedure Diagram

Figure 2. Illustration of the ChromaLINK Digoxigenin One-Shot antibody labeling procedure.

Process Summary

Sample Preparation: adjust antibody to 1 mg/ml in 100 μl buffer

Sample Analysis: confirm antibody concentration using a spectrophotometer

First Buffer Exchange: equilibrate spin columns & buffer exchange antibody

Sample Analysis: reconfirm recovered antibody concentration on a spectrophotometer

Digoxigenin Labeling: label antibody with ChromaLINK Digoxigenin linker

Second Buffer Exchange: remove excess labeling reagent with spin column

Digoxigenin Incorporation: quantify incorporation using a spectrophotometer

Important Labeling Parameters

The ChromaLINK Digoxigenin One-Shot antibody labeling kit is specifically designed to incorporate digoxigenin into a single 100 microgram quantity of antibody resuspended in 100 μl of a suitable buffer as indicated in Table 1.

Table 1. Initial starting conditions required for the ChromaLINK Digoxigenin One-Shot procedure.

The kit provides consistent and reliable incorporation of digoxigenin by controlling the following reaction variables:

Initial antibody mass (100 μg) and volume (100 μl)
Reaction buffer composition
Reaction time (60 min)
Reaction stoichiometry (15-fold mole-equivalents of linker)

Critical to digoxigenin incorporation is the ability to accurately determine an antibody’s initial concentration in a non-destructive manner with full recovery of the sample. The ChromaLINK Digoxigenin One-Shot procedure employs a spectral scan (220-400 nm) rather than a single wavelength measurement @ 280 nm to accomplish this task. Commercial antibodies often contain preservatives or other additives capable of masking or distorting the intrinsic UV spectrum of a sample. As a consequence, additives make it more difficult to accurately estimate protein concentration using a single wavelength (i.e. A280) measurement. A scan however, provides greater information and assurance that the antibody’s concentration is correct since a spectrum often reveals the presence of interfering additives.

Commonly used additives may include preservatives such as sodium azide, thimerosal, protein stabilizers such as *BSA or *gelatin, or small molecule additives such as glycine. *Note-protein carriers such as BSA or gelatin must be removed before labeling can proceed.

Legal Information

SoluLINK^® Bioconjugation For Research Use Only. Not for use in diagnostic procedures. For additional licensing restrictions, please see the license agreement at vectorlabs.com/solulink-research-license.

Products are for research use only, not for use in diagnostic or therapeutic procedures or for use in humans. Products are not for resale without express written permission of Seller. No license under any patent or other intellectual property right of Seller or its licensors is granted or implied by the purchase unless otherwise provided in writing.

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Protein–Oligo Conjugation Kit

Vector Laboratories R&D — Thu, 04 May 2023 04:57:33 +0000

Description

The Protein-Oligo Conjugation Kit is designed to easily and efficiently prepare two separate protein-oligo conjugates. This kit is flexible so that researchers with little or no conjugation experience can make their own custom protein-oligo conjugates to suit their research needs. It includes all of the necessary components, including S-HyNic and S-4FB, for the rapid and specific crosslinking of any protein with any oligo from 20 to 100 nucleotides in length.

The molecular weight range for proteins to be conjugated with this kit is 25,000 to 900,000 Daltons. Protein amounts from 50 – 650 µg at a concentration of 1 – 5 mg/mL in a volume of 50 – 130 µL are suitable for conjugation.

Specifications

Conjugation Targets	Protein
Label/Modifier Type	Oligonucleotide
Reactivity	Amine
Recommended Storage	2° – 8°C – Do Not Freeze
Applications	Antibody Labeling, In Situ Proximity Ligation

Kit Components

S-HyNic (2 X 1 mg)
S-4FB (2 x 1mg)
10X Modification Buffer (1.5 mL)
10X Conjugation Buffer (1.5 mL)
10X TurboLINK™ Catalyst Buffer (1.5 mL)
7kDa MWCO, 0.5 mL Thermo Scientific™ Zeba™ Column (4)
Anhydrous DMF (1.5 mL)
2-Hydrazinopyridine (2-HP) (0.5 mL)
2-Sulfobenzaldehyde (2-SB) (0.5 mL)
7kDa MWCO 2 mL Zeba™ Column (2)
10X PBS (1.5 mL)
2 mL Collection Tube (12)
Oligo Resuspension Solution (1.0 mL)

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Product FAQs

What is the difference between Protein-Oligo Conjugation Kit – (S-9011) and the Antibody – Oligonucleotide All-in-One Conjugation Kit (A-9202)?

The Protein-Oligo Conjugation Kit (S-9011) was designed with more flexibility in mind to allow conjugation of any protein greater than about 20 kDa, to an oligonucleotide. No purification material is included in this kit because there are so many different proteins that can be conjugated. This kit also allows for a variable amount of protein to be coupled (from about 50µg up to 650µg).

The Antibody – Oligonucleotide All-in-One Conjugation Kit (A-9202) was designed specifically for simple and reproducible coupling of an oligo to a fixed amount of antibody (100µg). All kit components have been optimized around this amount, which reduces the number of calculations involved and reduces variability in the process. This kit does include purification so the final conjugate is very pure (i.e., no free antibody or oligo remains in the final product), which allows kit usage in very sensitive assays. Since this kit is designed for antibodies, purification is accomplished using a resin that binds to all mammalian IgG antibody isotypes. Yields from this kit are typically 30-60% based on the amount of starting antibody. Longer oligos (60-mers and up) tend to have a lower yield (in the 20-40% range) due to steric hinderance of the antibody binding to the resin.

Citations

Technical Information

I. Introduction

a. Product Description

The Protein-Oligo Conjugation Kit is designed to conjugate a protein with an oligonucleotide. It includes all of the necessary components and protocols for easy and specific crosslinking of any protein with any amino-oligo from 20 to 100 bases in length. This kit is flexible so that researchers with little or no conjugation experience can make their own custom protein-oligo conjugate to suit their needs.

SoluLINK bioconjugation technology converts more than 95% of protein to conjugate when four mole equivalents of oligo are added. High conversion rates, coupled with the unique UV-traceable bond formed during crosslinking, allows for easy purification and identification of the conjugate from the excess oligo using size exclusion purification methods such as HPLC.

b. SoluLINK Bioconjugation Technology

The Protein-Oligo Conjugation Kit uses the superior SoluLINK bioconjugation technology to prepare protein-oligonucleotide conjugates in 3 easy-to-perform steps (Figure 1). The first step is the modification of the oligo with our 4FB crosslinker, followed by the formation of the HyNic modified protein. Finally, simple mixing of the two modified biomolecules will result in the formation of a stable, UV-traceable bond formed by the reaction of a HyNic modified protein with a 4FB modified oligonucleotide.

This technology has many practical advantages compared to previous crosslinking methods:

1. The reaction is high yielding. Routine yields of conjugate are 50-80% based on starting protein.

2. The reaction is efficient: Only 3-4 mole equivalents of oligo are necessary for the protein, >90% of the protein is conjugated.

3. The conjugate bond is extremely stable: The conjugate bond is stable to 92℃ and pH 2.0-10.0.

4. The reaction conditions are mild and do not cause any protein denaturation: unlike thiol-based conjugation protocols, where reducing reagents are required that can compromise the activity of proteins by cleaving disulfide bonds, the HyNic-4FB conjugation couple leaves disulfide bonds intact. No metals, oxidation or reducing reagents are required.

Figure 1: Schematic representation of the three step process to prepare an antibody-oligonucleotide conjugate using SoluLINK Bioconjugation technology. Initially an antibody is modified with S-HyNic to incorporate HyNic groups and subsequently the HyNic-modified antibody is reacted with a 4FB-modified oligonucleotide.

5. The conjugation is traceable spectrophotometrically. The HyNic-4FB conjugate bond is UV-traceable; it absorbs at 354 nm and has a molar extinction coefficient of 29,000 L/(mol*cm).

6. The modifications of both the HyNic linker on the protein and the 4FB linker on the oligonucleotide are quantifiable using colorimetric assays. The reproducibility of any reaction is facilitated by accurate characterization of all components. The Molar Substitution Ratio (MSR) of linker groups, i.e. the number of HyNic linkers per protein, can be quantified colorimetrically. This kit contains all the reagents necessary to determine the MSRs for both the protein and the oligo.

II. Protein-Oligonucleotide Conjugates: A Review

The diversity and specificity of proteins combined with the specificity of hybridization of oligonucleotides results in unlimited numbers of specific protein detection reagents whose applications are addressed below.

The use of oligo-protein conjugates was initially demonstrated by Sano et al. for protein detection by a technique called immuno-PCR (Polymerase Chain Reaction) where a 100-mer oligo/antibody conjugate was allowed to bind to its ligand and amplified by PCR demonstrating extremely sensitive protein detection. Since this initial publication there has been a need for a straight forward, efficient and high yielding method for the preparation of these conjugates.

The first generation immuno-PCR protocol was plagued by high background due to non-specific binding of the conjugate and the extreme sensitivity of PCR. This has been overcome by the Proximal Ligation Assay (PLA) developed by Fredriksson and Lundegren. In the PLA assay, two antibodies to different epitopes are conjugated to a 40-mer 5’-phosphorylated oligonucleotide through the 3’-end and 60-mer oligonucleotide conjugated through its 5’-terminus. The two oligo/antibody conjugates are incubated with the sample, allowed to bind to their respective epitopes, the mixture is washed and then incubated with a ‘splint’ oligo that hybridizes across the two oligonucleotides that is subsequently ligated. Following ligation, PCR is performed on the ligated oligo generating a quantifiable signal. In subsequent work the oligo/antibody conjugates used by Fredriksson et al. and others used conjugates using the HyNic-4FB conjugation method. Kozlov et al also describe the use of oligonucleotide/antibody conjugates for the sensitive detection of proteins.

Additionally, oligonucleotide/antibody conjugates have been used for capture of antigens and subsequent addressing to antibody arrays for multiplex detection of proteins as well for cell sorting on the same diagnostic platform. Oligonucleotide/protein conjugates have been also been used in vaccines to increase adjuvanticity using CpG oligonucleotide/protein conjugates.

III. Accessing 4FB-Modified Oligonucleotides

Stable and disulfide-cleavable 4FB oligonucleotides can be obtained in several ways:

5’-4FB oligonucleotide
a. 4FB-phosphoramidite: 4FB-Phosphoramidite (1; Figure 2) is available for incorporation of 5’-4FB groups during oligonucleotide solid phase synthesis. Standard coupling protocols are used and the yields are similar to any amino modifier.
b. 5’-amino oligonucleotides: 5’-amino oligonucleotides may be converted to 5’-4FB modified oligonucleotides in a straightforward high yielding modification step with S-4FB (2; Figure 2).
The Protein-Oligo Conjugation Kit includes S-4FB and all the reagents and materials required to convert a 5’-amino oligonucleotide to a 5’-4FB-oligonucleotide.
3’-4FB oligonucleotide:
3’-Amino oligonucleotides are converted to 3’-4FB modified oligonucleotides in an easy, high yielding modification step with S-4FB (2; Figure 2).
The Protein-Oligo Conjugation Kit includes S-4FB and all the reagents and disposables required to convert a 3’-amino oligonucleotide to a 3’-4FB-oligonucleotide.
5’- and 3’-4FB disulfide-cleavable oligonucleotides: 5’- and 3’-amino oligonucleotides may be converted to disulfide-cleavable oligonucleotides using S-SS-4FB (3; Figure 2) in an easy, high yielding modification step.

Figure 2: Schematic representation of the conversion of an amino-modified oligonucleotide to a 4FB-oligonucleotide with S-4FB (2) (top) and structures of 4FB-phosphoramidite (1) and S-SS-4FB (3), the reagent used to convert an amino-oligonucleotide to a 4FB-SS-oligonucleotide.

IV. The Keys to Successful Conjugation

The following are three crucial requirements that must be fulfilled for a reproducibly successful preparation of a protein-oligonucleotide conjugate using the SoluLINK bioconjugation technology:

Desalting: Prior to modification, the starting protein must be thoroughly desalted, removing all amine contaminants, and exchanged into 1X Modification Buffer.
Protein concentration: The recommended concentration of the protein (1–5mg/ml) must be adhered to in all steps.
Molar substitution ratio: The molar ratio of the HyNic on the protein and the 4FB on the oligo must be determined and within the desired range before continuing to the next step.

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Zeba and Thermo Scientific are trademarks of Thermo Fisher Scientific Inc.

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Protein–Protein Conjugation Kit

Vector Laboratories R&D — Thu, 04 May 2023 04:05:06 +0000

Description

The Protein-Protein Conjugation Kit is designed to easily and efficiently conjugate any two proteins. This kit is flexible so that researchers with little or no bioconjugation experience can make their own custom protein-protein conjugates to suit their research needs. It includes all of the necessary components, including S-HyNic and S-4FB, for the rapid and specific crosslinking of any two proteins.

The molecular weight range for proteins to be conjugated with this kit is 25,000 to 900,000 Daltons. to The molecular weight range for proteins to be conjugated with this kit is 25–950 kDa.

Specifications

Conjugation Targets	Protein
Label/Modifier Type	Protein
Reactivity	Amine
Recommended Storage	2° – 8°C – Do Not Freeze
Applications	Antibody Labeling, In Situ Proximity Ligation

Kit Components

S-HyNic (2 x 1.0 mg)
S-4FB (2 x 1.0 mg)
10X Modification Buffer (1.5 mL)
10X Conjugation Buffer (1.5 mL)
10X TurboLINK™ Catalyst Buffer (1.5 mL)
7kDa, 0.5 mL Thermo Scientific™ Zeba™ Column (10)
Anhydrous DMF (2 x 1.5 mL)
2-Hydrazinopyridine (2-HP) reagent (0.5 mL)
0.5mM 2-Sulfobenzaldehyde (2-SB) (0.5 mL)
2 mL Collection Tube (10)
7kDa, 2 mL Zeba™ Column (2)
10X PBS (1.5 mL)

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Technical Information

The ChromaLINK Reaction:

ChromaLINK bioconjugation technology is based on the formation of a stable bis-arylhydrazone formed from an aromatic hydrazine and an aromatic aldehyde. S-HyNic (succinimidyl 6-hydrazinonicotinate acetone hydrazone, SANH) is used to incorporate aromatic hydrazine moieties on biomolecules. S-HyNic is an amine-reactive reagent that directly converts amino groups on biomolecules and surfaces to HyNic groups. S-4FB (succinimidyl 4-formylbenzoate, SFB) is used to convert amino groups to aromatic aldehydes (4-formylbenzamide (4FB) groups). Addition of a HyNic-modified biomolecule to a 4FB-modified biomolecule or surface directly leads to the formation of the conjugate (Figure 1). The bis-arylhydrazone bond is stable up to 92℃ and pH 2.0-10.0. Due to lability of the immunoreactivity of antibodies at low pH, i.e. < 5.0, the recommended pH for antibody conjugation is 6.0. Unlike thiol-based conjugation protocols where reducing reagents are required that can compromise the activity of proteins by cleaving disulfide bonds, the HyNic-4FB conjugation couple leaves disulfide bonds intact. No oxidants, reducing agents, or metals are required in the preparation of conjugate.

Figure 1: Linking chemistry behind ChromaLINK bioconjugation technology.

Fastest, most efficient:

Further enhancing the many advantages of the HyNic/4FB conjugation couple is the discovery by Dirksen et al. that showed aniline catalyzes the formation of this Schiff’s base. This is especially effective for large biomolecule conjugations. In the case of antibody-protein conjugations the addition of 10 mM aniline to the reaction mixture converts >95% of the antibody to conjugate in 2 hours using 1-2 mole equivalents of second protein.

Traceable modification:

Reproducibility of any reaction is dependent on accurate characterization of all components. As both HyNic and 4FB are aromatic, their incorporation can be readily quantified using colorimetric assays.

Traceable conjugation:

The HyNic-4FB conjugate bond is chromophoric. It absorbs light at 354 nm and has a molar extinction coefficient of 29,000 L/(mol*cm).

Furthermore, compared to previous methods, the HyNic/4FB technology offers the following practical advantages:

1) The reaction goes to completion: In all previous bi-functional linker-based conjugations, the reaction never went to completion, i.e. there was always unconjugated limiting protein in the final product. The HyNic-4FB conjugation couple catalyzed by aniline yields more than 95% conjugate.

2) The reaction is efficient: The reaction is very stoichiometrically efficient as input of only 1-2 moles of second protein/mole first protein is required for complete conversion to conjugate.

3) The conjugate bond is extremely stable: the bis-arylhydrazone conjugate bond is stable up to 92℃ and pH 2.0-10.0.

4) The reaction conditions are extremely mild and do not cause antibody denaturation: unlike thiol-based conjugation protocols where reducing reagents are required that can compromise the activity of proteins by cleaving disulfide bonds, the HyNic-4FB conjugation couple leaves disulfide bonds intact. No metals, oxidizing, or reducing reagents are required.

5) The conjugation is traceable spectrophotometrically: the HyNic-4FB conjugate bond is chromophoric; it absorbs light at 354 nm and has a molar extinction coefficient of 29,000 L/(mol*cm).

6) The modifications of both the HyNic moiety on the protein and the 4FB moiety on the protein is quantifiable using a colorimetric assay: the reproducibility of any reaction is dependent on accurate characterization of all components. The Molar Substitution Ratio (MSR; i.e. the number of HyNic groups incorporated per protein) can be quantified colorimetrically as a reaction with 2-sulfobenzaldehyde yields a chromophoric product that absorbs at 350 nm with a molar extinction coefficient of 28,500 L/(mol*cm). The MSR of 4FB groups can be determined colorimetrically by its reaction with 2-hydrazinopyridine forming a hydrazone that absorbs at 348 nm with a molar extinction coefficient of 24,500 L/(mol*cm). This kit contains all the reagents necessary to determine both MSRs. Procedures to guide users through this process are given in the protocol below.

The Keys to Successful Conjugation

The following are three crucial requirements that must be fulfilled for a reproducibly successful preparation of a protein-protein conjugate using the SoluLINK bioconjugation technology:

1. Desalting: prior to modification, the starting proteins must be thoroughly desalted, removing all amine contaminants and exchanging the proteins into 1X Modification Buffer.

2. Protein concentration: the recommended protein concentrations must be adhered to in all steps.

3. Molar substitution ratio: the molar ratio of HyNic on the protein and 4FB on the protein must be determined and within the desired range before continuing to the next step.

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Zeba and Thermo Scientific are trademarks of Thermo Fisher Scientific Inc.

The post Protein–Protein Conjugation Kit appeared first on VectorLabs.

Conjugation Buffer (10X)

Vector Laboratories R&D — Thu, 04 May 2023 03:58:24 +0000

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Description

Conjugation Buffer is a high buffering capacity solution that is optimized for the conjugation reaction between HyNic and 4FB groups. Proteins, oligonucleotides, and other biomolecules are buffer exchanged into Conjugation Buffer after modification with S-HyNic and S-4FB, and prior to conjugation, to ensure efficient conjugation of these linkers.

Specifications


Recommended Storage	2° – 8°C – Do Not Freeze
Applications	In Situ Proximity Ligation
Other Name(s)	SoluLINK Bioconjugation

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Anhydrous DMF

Vector Laboratories R&D — Thu, 04 May 2023 03:52:35 +0000

Description
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Description

Anhydrous DMF (Dimethylformamide) is used for solubilizing S-HyNic, S-4FB, and other SoluLINK linkers. Anhydrous DMF is packaged with molecular sieves to minimize water contamination and preserve quality.

Specifications

Recommended Storage	15° – 25°C
Applications	In Situ Proximity Ligation
Other Name(s)	Dimethylformamide SoluLINK Bioconjugation

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Modification Buffer (10X)

Vector Laboratories R&D — Wed, 03 May 2023 21:45:22 +0000

Description
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Description

Modification Buffer is an amine-free, high buffering capacity solution which is optimized for the reaction of NHS esters and TFP esters with amino groups. Proteins, oligonucleotides, and other biomolecules are buffer exchanged into Modification Buffer prior to modification with S-HyNic and S-4FB to ensure efficient coupling of these linkers.

Specifications

Recommended Storage	2° – 8°C – Do Not Freeze
Applications	In Situ Proximity Ligation
Other Name(s)	SoluLINK Bioconjugation

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10X TurboLink™ Catalyst Buffer

Vector Laboratories R&D — Wed, 03 May 2023 21:40:43 +0000

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Description

TurboLINK Catalyst Buffer is used to catalyze the SoluLINK^® bioconjugation reactions, which are based on aromatic hydrazone formation between HyNic-modified biomolecules and 4FB-modified biomolecules. TurboLINK greatly accelerates hydrazone formation — in the presence of TurboLINK Catalyst the conjugation reaction is typically complete in 1-2 hours. The active ingredient, aniline, is delivered at 100 mM concentration in Conjugation Buffer. A final concentration of 5-10 mM aniline in the conjugation reaction is sufficient for hydrazone catalysis. This reagent is particularly useful for conjugation of large biomolecules such as antibodies, oligonucleotides, proteins, enzymes, and surfaces.

Specifications


Recommended Storage	2° – 8°C – Do Not Freeze
Applications	In Situ Proximity Ligation
Other Name(s)	SoluLINK Bioconjugation

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R-PE Antibody Conjugation Kit

Vector Laboratories R&D — Wed, 03 May 2023 20:50:47 +0000

Description

The R-PE Antibody Conjugation Kit is a flexible alternative to the R-PE Antibody All-in-One™ Conjugation Kit. Each kit contains sufficient material for two reactions of up to 1.3 mg antibody each. Based on SoluLINK^® bioconjugation technology, the reactions are rapid and high-yielding, converting nearly 100% of antibody to PE conjugate. This kit is ideal for applications where purification to remove excess R-PE is either not required, or purification will be carried out by chromatographic methods (such as size exclusion FPLC).

Specifications


Conjugation Targets	Antibody
Label/Modifier Type	R-PE
Reactivity	Amine
Recommended Storage	2° – 8° C — Do Not Freeze
Applications	Antibody Labeling

Kit Components

S-HyNic (2 x 1.0 mg)
4FB-R-PE (2 x 3.2 mg)
10X Modification Buffer (1.5 mL)
10X Conjugation Buffer (1.5 mL)
10X TurboLINK™ Catalyst Buffer (1.5 mL)
Anhydrous DMF (1.5 mL)
0.5 mL Thermo Scientific™ Zeba™ Column (4)
2 mL Collection Tube (12)
2-Sulfobenzaldehyde, 2-SB (10 mg)
10X MES Buffer (1.5 mL)
2 mL Zeba™ Column (2

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Technical Information

R-PE-Antibody Conjugation Kit Using the SoluLINK bioconjugation Technology

The SoluLINK technology can be used to more easily and efficiently prepare R-PE-antibody conjugates as compared to alternative (maleimide/thiol-based) protocols. Other conjugation methods expose thiols on antibodies by DTT reduction of disulfide bonds, which cleaves the antibody into a variety of species. The SoluLINK bioconjugation technology, however, gently incorporates HyNic moieties on the intact antibody.

This technology is superior to the maleimide/thiol-based method in the following ways:

Efficiency: Greater than 95% of antibody is converted to conjugate and only 1-1.5 molar equivalents of R-PE is required per mole of antibody to produce the conjugate.
Ease of purification: In most cases, the percent conversion of free antibody to conjugate is >95. Therefore it is only necessary to remove the excess R-PE to obtain a purified conjugate and in many applications, purification is not necessary.
Reaction Control: The level of polymerization, and therefore the brightness of the conjugate, can be controlled by adjusting the level of HyNic modification on the antibody. This is particularly helpful in some applications, such as flow cytometry, where a heterodimer product is preferred.

Introduction to SoluLINK Bioconjugation Technology

The SoluLINK bioconjugation technology is based on the formation of a stable bond formed by the reaction of an aromatic hydrazine and an aromatic aldehyde (Figure 1). S-HyNic is an amino-reactive modification reagent that directly converts amino groups on biomolecules and surfaces to HyNic groups. S-HyNic 1 (succinimidyl 6-hydrazinonicotinate acetone hydrazone, SANH) is used to incorporate aromatic hydrazine linkers on biomolecules. S- 4FB 2 (succinimidyl 4-formylbenzoate, SFB) is used to convert amino groups to aromatic aldehydes (4-formylbenzamide (4FB) groups). Addition of a HyNic-modified biomolecule to a 4FB-modified R-PE leads to the formation of the conjugate via a bis-arylhydrazone bond. The bis-aryl hydrazone bond is stable to 92°C and pH 2.0-10.0. The recommended pH for antibody conjugation is 6.0.

Figure 1: Schematic of the two-step protocol to prepare R-PE-antibody conjugates. Step 1 is the modification of antibody with S-HyNic to prepare HyNic-antibody. Step 2 is the conjugation of HyNic-antibody to 4FB-R-PE by mixing in 100 mM phosphate, 150 mM NaCl, 10 mM aniline, pH 6.0.

Advantages of the HyNic-4FB conjugation couple include:

High Yielding: Yields of conjugate are routinely 40-60%, based on starting protein.
Efficient: Dirksen et al^* discovered that aniline catalyzes Schiff’s base formation. This is especially effective for large biomolecule conjugations. In the case of antibody-protein conjugations, the addition of 10 mM aniline to the reaction mixture converts >95% of the antibody to conjugate in ~2 hours using 1-1.5 mole equivalents of second protein as strikingly demonstrated in this product.
Stability: The bis-arylhydrazone conjugate bond is stable to 92°C and pH 2.0-10.0.
Mild reaction conditions: These mild conditions do not cause antibody denaturation: Unlike thiol-based conjugation protocols where reducing reagents are required that can compromise the activity of proteins by cleaving disulfide bonds, the HyNic-4FB conjugation couple leaves disulfide bonds intact. No metals, oxidation or reducing reagents are required.
Spectrophotometric Traceability: The HyNic-4FB conjugate bond is chromophoric, it absorbs at 354 nm and has a molar extinction coefficient of 29,000 L/(mol*cm). This allows,
- Real time spectrophotometric monitoring of a conjugate reaction.
- The ability to visualize the conjugate during chromatographic purification using a UV or photodiode array detector.
- Quantification of conjugation.

There are three crucial requirements that must be fulfilled for a reproducibly successful preparation of an R-PE Antibody conjugate using the SoluLINK bioconjugation technology:

Antibody buffer exchange: Prior to modification, the starting antibody must be completely exchanged into Modification Buffer, pH 8.0.
Minimum level of HyNic-modification: The HyNic-antibody molar substitution ratio (MSR) must be >3.5 as determined by a colorimetric assay.
HyNic Concentration: The final concentration of the HyNic-antibody in the conjugation reaction must be >1.5 mg/mL.

References

^*Dirksen, A., et al. Nucleophilic catalysis of hydrazone formation and transimination: implications for dynamic covalent chemistry. J Am Chem Soc, 2006. 128(49): p. 15602‐3.

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Zeba and Thermo Scientific are trademarks of Thermo Fisher Scientific Inc.

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ChromaLINK® Biotin Protein Labeling

Vector Laboratories R&D — Wed, 03 May 2023 04:40:32 +0000

Description

The ChromaLINK Biotin Protein Labeling Kit includes everything you need to biotinylate your protein: buffers, reagents, spin columns, and an easy-to-follow protocol and online calculator. With the ChromaLINK Biotin Protein Labeling Kit you can biotinylate and purify up to 5 antibodies or other proteins ranging in molecular weight from 20–200 kDa and from as little as 25 µg to as much as 1 mg.

Specifications

Conjugation Targets	Antibody
Label/Modifier Type	biotin
Reactivity	Amine
Recommended Storage	2° – 8°C — Do Not Freeze
Applications	Antibody Labeling

Kit Components

ChromaLINK Biotin (5 x 0.5 mg)
10X Modification Buffer (2 x 1.5 mL)
10X PBS (2 x 1.5 mL)
0.5 mL Zeba™ Column (12)
2 mL Collection Tube (24)
1.5 mL Storage Tube (12)
Bovine IgG Control (0.5 mg)
Biotinylated Bovine IgG Control (0.5 mg)
Anhydrous DMF (1.5 mL)

Documents

Technical Information

A. Product Description

The ChromaLINK Biotin Labeling kit has all the necessary reagents for the traceable biotinylation of any lysine-containing protein. This kit provides sufficient materials to biotinylate and purify up to 5 proteins in about 2 hours. The scale of each protein labeling reaction is chosen by the researcher and can span from as little as 25 μg to as much as 1 milligram of protein per reaction.

The ChromaLINK Biotin labeling process is based on a patented UV-traceable linker called ChromaLINK Biotin (Figure 1). This process is based on an advanced protein biotinylation reagent containing an aromatic, N-hydroxy-succinimidyl ester functional group (a), which efficiently modifies protein lysine residues in a phosphate buffer system. The linker possesses an embedded bis-aryl hydrazone structure (b), which forms the compound’s UV-traceable chromophore. The traceable nature of this unique chromophore enables the non-destructive quantification of biotin (c), attached to the protein. These three diverse chemical elements are linked together through a long-chain PEG3 spacer (d), which preserves streptavidin/biotin affinity while simultaneously maintaining antibody solubility.

Figure 1. Structure of ChromaLINK Biotin C38H50N8O10S; MW: 810.92

b. Benefits and Features

The ChromaLINK Biotin Labeling kit offers an easier solution to the quantitative biotinylation of proteins starting from as little as 25 μg to as much as 1 milligram. Proteins ranging in molecular weights from 20–200 kDa are readily biotinylated using ChromaLINK Biotin. The accompanying instructions and protocols are designed to make traceable biotinylation of proteins easy to perform. The use of ChromaLINK Biotin eliminates the need to carry out cumbersome and indirect biotin binding assays such as the time consuming HABA or FluoroReporter™ assay used to determine the degree of biotin incorporation. Rather, after labeling, the biotin molar substitution ratio is measured directly by a simple UV-spectrophotometric scan (220–420 nm) or single point measurements at two wavelengths (A280/A354). Alternatively, a single wavelength (A354) can be used in combination with a protein assay (e.g., BCA or Bradford) to quantify the molar substitution ratio (MSR) or number of biotins per protein molecule.

c. Protocol Summary

Sample preparation (10 min)
1st buffer exchange (10 min)
Protein determination assay (BCA assay) (30 min)
Biotinylation (120 min)
2nd buffer exchange (10 min)
UV analysis and calculations (20 min)

d. Labeling Process Diagram

The ChromaLink Biotin protein labeling process is diagramed in Figure 2.

Figure 2. ChromaLink Biotin protein labeling process.

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ChromaLINK® One-Shot™ Antibody Biotinylation Kit

Vector Laboratories R&D — Wed, 03 May 2023 04:32:38 +0000

Description

The ChromaLINK One-Shot Antibody Biotinylation Kit provides convenient, consistent, and measurable biotinylation of 100 µg of antibody. Each kit contains ChromaLINK Biotin, which incorporates a novel UV-traceable chromophore in the linker arm to enable reproducibility in your biotinylation process. Now you can measure the degree of biotinylation in minutes, not hours, without the standard curves required for the HABA/Avidin and Fluoro-Reporter assays. The ChromaLINK One-Shot kit contains everything needed to to biotinylate your antibody: buffers, reagents, desalting columns, and an easy-to-follow protocol and online biotin incorporation calculator.

Specifications

Conjugation Targets	Antibody
Label/Modifier Type	biotin
Reactivity	Amine
Recommended Storage	2° – 8°C — Do Not Freeze
Applications	Antibody Labeling, In Situ Proximity Ligation
Unit Size	1 Kit

Kit Components

ChromaLINK Biotin (6.49 μg)
1X Modification Buffer (1.5 mL)
1X PBS (2 x 1.5 mL)
2 mL Collection Tube (4)
0.5 mL Zeba™ Desalting Column (2)
Biotinylated Bovine IgG Control (100 μg)
1M Tris HCI (1.5 mL)
Anhydrous DMF (1.5 mL)

Documents

Product FAQs

For B-9007-105K, why you would need to perform buffer exchange on the protein if you have reconstituted it in the 1x modification buffer?

The purpose of the buffer exchange step is to remove amine contaminants, such as Tris or glycine, from the protein. These contaminants may interfere with NHS ester labeling, which targets primary amine groups. Some samples are so excessively contaminated that two buffer exchanges may be required before proceeding with biotinylation.

Citations

Technical Information

A. Product Description

The ChromaLINK Biotin One-Shot Antibody Labeling Kit is designed to biotinylate a single 100 microgram quantity of antibody in about 2.0 hours. The ChromaLINK One-Shot kit relies on a UV-traceable linker called ChromaLINK Biotin to label the antibody (Figure 1). This unique labeling reagent contains an aromatic, water-soluble N-hydroxy-succinimidyl ester functional group. (a) that efficiently modifies antibody lysine residues under mild phosphate buffer conditions. The reagent also possesses an embedded bis-aryl hydrazone structure (b) which forms the linker’s UV-traceable chromophore. The absorbance signature provided by the chromophore enables rapid and non-destructive quantification of incorporated biotin. (c). The linker also features a long PEG3 spacer (d) which preserves streptavidin/biotin affinity and helps maintain antibody solubility.

Figure 1. Structure of ChromaLINK Biotin C38H50N8O10S; MW 810.92.

B. Features and Benefits

The ChromaLINK Biotin One-Shot Kit is a simple, cost-effective way of incorporating a readily verifiable amount of biotin into a single 100 μg quantity of antibody. The kit conveniently features a pre-weighed amount of ChromaLINK Biotin reagent that is readily resuspended directly into the antibody solution. ChromaLINK Biotin can be used to label a variety of different antibodies, including mammalian IgG (monoclonal or polyclonal) and/or avian IgY. Biotin incorporation is rapidly determined by means of a simple, non-destructive UV-scan (220–400 nm) of the sample after removal of excess labeling reagent. The kit features high antibody recovery (60–90 μg) and a consistent level of biotin incorporation (3–8 biotin molecules per antibody molecule) when used as directed.

C. ChromaLINK One-Shot Process Diagram

The ChromaLINK Biotin One-Shot antibody labeling procedure is illustrated in Figure 2.

Figure 2. ChromaLINK Biotin One-Shot antibody labeling procedure.

D. Process Summary

Sample Preparation: bring antibody to 1 mg/mL in 100 μL buffer
1st Sample Analysis: confirm antibody concentration using spectrophotometer
1st Buffer Exchange: equilibrate spin columns and buffer exchange antibody
2nd Sample Analysis: reconfirm antibody concentration using spectrophotometer
Biotinylation: label antibody with ChromaLINK Biotin
2nd Buffer Exchange: remove excess labeling reagent
Quantify Biotin Incorporation: measure biotin incorporation using spectrophotometer

E. Important Labeling Parameters

The ChromaLINK Biotin One-Shot Antibody Labeling Kit is designed to biotinylate a single 100 μg quantity of antibody resuspended in 100 μL buffer as indicated in Table 1.

Table 1. Initial conditions required for the ChromaLINK Biotin One-Shot labeling procedure.
This kit provides a consistent and reliable degree of biotinylation by controlling the following reaction variables:

Fixed antibody mass (100 μg) and volume (100 μL)
Optimized reaction buffer
Controlled and fixed reaction time (90 min)
Consistent reaction stoichiometry (12X mole-equivalents) (12-fold molar excess of biotin reagent)

Critical to consistent biotin incorporation is the ability of a user to accurately determine the antibody’s initial protein concentration in a non-destructive manner that allows full or nearly full recovery of the precious sample. The ChromaLINK One-Shot procedure recommends using a scan (220–400 nm) on a spectrophotometer rather than the traditional A280 to estimate antibody concentration. The rationale for using a scan rather than a single wavelength (A280) is that many commercial antibodies contain preservatives and/or other additives that mask or distort the intrinsic A280 of a sample. The presence of an additive can make it more difficult or sometimes impossible to accurately estimate protein concentration via A280. A scan however contains greater spectral information, often revealing the presence of spectrum-altering additives that are known to interfere with the accurate estimate of protein concentration. Altered spectra are a warning that the A280 of the sample will not yield an accurate estimate of protein concentration. Commonly used additives include preservatives such as sodium azide, thimerosal, protein stabilizers such as BSA or gelatin and/or small molecule additives such as glycine or trehalose.

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