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

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)

Documents

• User Guide
• Bioconjugation White Paper
• Safety Data Sheet
• S-9011 – Protein-Oligonucleotide Conjugation Calculator
• Troubleshooting Guide – Bioconjugation
• BCA Protein Assay Protocol
• Bradford Assay Protocol
• Download CoA
• Datasheet

Product FAQs

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.

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.

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

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

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)

Documents

• User Guide
• Bioconjugation White Paper
• Safety Data Sheet
• P-9002 – Antibody / R-Phycoerythrin Conjugation Calculator
• Troubleshooting Guide – Bioconjugation
• BCA Protein Assay Protocol
• Bradford Assay Protocol
• Download CoA
• Datasheet

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.

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.

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

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

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

• User Guide
• Safety Data Sheet
• ChromaLINK Biotin Conjugation Calculator
• Troubleshooting Guide – Bioconjugation
• Download CoA
• Datasheet

Product FAQs

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

1. Sample Preparation: bring antibody to 1 mg/mL in 100 μL buffer
2. 1st Sample Analysis: confirm antibody concentration using spectrophotometer
3. 1st Buffer Exchange: equilibrate spin columns and buffer exchange antibody
4. 2nd Sample Analysis: reconfirm antibody concentration using spectrophotometer
5. Biotinylation: label antibody with ChromaLINK Biotin
6. 2nd Buffer Exchange: remove excess labeling reagent
7. 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.

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.

Description

The Antibody-Oligonucleotide All-in-One Conjugation Kit uses patented SoluLINK^® bioconjugation technology to link an antibody to an oligonucleotide. A succinimidyl-4-formylbenzamide (S-4FB)-modified oligonucleotide and succinimidyl-6-hydrazino-nicotinamide (S-HyNic)-modified antibody form an antibody-oligonucleotide conjugate. This kit is specifically designed to conjugate 100 µg of antibody to any amino-modified oligonucleotide in the 20-80 nucleotide range.

Key Features:

• Any mammalian monoclonal or polyclonal IgG isotype antibody can be conjugated to the oligonucleotide and purified with approximately 4 hours of total hands-on time.
• SoluLINK bioconjugation technology forms a stable and UV-traceable bis-aryl hydrazone bond with measurable absorbance at 354 nm, which has a molar extinction coefficient of 29,000 L mol^-1 cm^-1.
• The conjugation reaction is spectrophotometrically quantifiable due to the formation of a chromophoric conjugate bond.

Specifications

Kit Components

Documents

• User Guide
• A-9202-001, Antibody–Oligonucleotide Conjugation Calculator
• Bioconjugation White Paper
• Safety Data Sheet
• Troubleshooting Guide – Bioconjugation
• Concentration of Dilute Antibody Solutions
• Download CoA
• Datasheet

Product FAQs

Technical Information

Antibody-Oligonucleotide All-in-One Conjugation Kit

Overview of Conjugation

1) Conjugation Chemistry

The Antibody-Oligonucleotide All-in-One Conjugation kit uses the SoluLINK patented conjugation technology to link an antibody to an oligonucleotide, as illustrated in Figure 1. The first stage of the process begins with the labeling of a 3’ or 5’- amino-modified oligonucleotide withS-4FB. This amine-reactive NHS ester incorporates an aromatic aldehyde functional group, formylbenzamide (4FB), at the desired terminus of the oligonucleotide.

Figure 1. Reaction of a HyNic-modified IgG with a 4FB-modified oligo leads to the rapid formation of a stable antibody-oligonucleotide conjugate.

In stage two of the process, a polyclonal or monoclonal antibody (100 μg) is modified using the complementary linker S-HyNic. This NHS-ester reacts with lysine residues, incorporating HyNic functional groups (hydrazinonicotinamide) onto the antibody. In the third and final stage, the two modified biomolecules are mixed together in the presence of a reaction catalyst (aniline) to form the conjugate, after which purification is carried out using a magnetic affinity solid phase.

2) Conjugate Purification

Antibody-oligonucleotide conjugates produced with the All-in-One kit are ready to be used in the most demanding and sensitive of downstream applications. The kit delivers high-purity conjugate virtually free of residual antibody or oligonucleotide (>98%). Reaction conditions are optimized to convert nearly 100% of the antibody into conjugate, leaving only free, excess 4FB-oligo to be removed. Complete conversion of antibody to conjugate simplifies conjugate purification as illustrated in Figure 2. Antibody-oligonucleotide conjugate is purified to near homogeneity by selectively binding the conjugate to a magnetic affinity matrix, allowing excess 4FB-oligonucleotide to be washed away. Affinity-bound conjugate is then gently eluted from the matrix and buffer exchanged into long-term storage buffer. Antibody-oligonucleotide conjugates produced with this kit are stable for up to 1 year when kept at 4^oC in storage buffer.

Figure 2. All-in-One conjugate purification strategy.

All-in-One Conjugation Overview

* 4FB MSR is an acronym for 4-formylbenzamide molar substitution ratio.

Figure 3. Stage 1 of the process illustrates the modification of an amino-oligonucleotide with the S-4FB linker.

Figure 4. Stage 2 of the process illustrates the modification of IgG using the S-HyNic linker.

Figure 5. Stage 3 of the process illustrates both the formation and purification of the conjugate.

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.

Description

The HRP-antibody All-in-One Conjugation Kit contains everything needed to make two HRP-antibody conjugates from 100 µg of any user-supplied antibody. Included in the kit are purification columns to remove unconjugated HRP and antibody, which produces conjugates with superior levels of detection and low non-specific binding.

Specifications

Kit Components

• S-HyNic (2 x 100 μg)
• 4FB-HRP (2 x 100 μl)
• 1X Modification Buffer (10 mL)
• Q Binding Buffer A (5 mL)
• Q Elution Buffer A (1.6 mL)
• Red Cap Spin Column (2)
• Yellow Cap Spin Column B (2)
• Brown Cap Spin Column B (2)
• Blue Cap Spin Column B (2)
• Q Spin Column (2)
• Q Collection Tube (4)
• Anhydrous DMF (1.5 mL)
• 30K MWCO VivaSpin^® Filter (2)
• 2 mL Collection Tube (16)

Documents

• User Guide
• Bioconjugation White Paper
• Safety Data Sheet
• Troubleshooting Guide – Bioconjugation
• Concentration of Dilute Antibody Solutions
• Using a NanoDrop to Measure Antibody Concentration
• BCA Protein Assay Protocol
• Bradford Assay Protocol
• Download CoA
• Datasheet

Technical Information

A. Product Description

Each HRP‐antibody All‐in‐One Conjugation Kit provides all the necessary components to generate two (2) highly purified antibody-HRP conjugates. The kit requires the user to provide 100 μg of starting antibody for each conjugate. The components of this unique kit feature a pre‐activated, high‐activity horseradish peroxidase (>250U/mg) as well as a novel Q spin filter to purify the conjugate in high yield. Conjugates produced are free of both residual antibody and HRP, thus providing maximum signal to noise ratio in your assay. Any suitably purified monoclonal or polyclonal mammalian antibody (regardless of IgG subclass) can be conjugated and purified within 5 hours (~1 hour hands‐on).

All‐in‐One conjugation kits are based on the SoluLINK bioconjugation technology. This chemistry involves the reaction of an aromatic hydrazine with an aromatic aldehyde to form a stable hydrazone bond. This conjugation is so efficient that it converts 100% of the antibody to the conjugate form. This linking efficiency is made possible because of the recent discovery that small quantities of aniline catalyze hydrazone bond formation between the two functional groups (1, 2, 3). Aniline increases both the rate and efficiency of conjugate formation under mild reaction conditions, leading to quantitative conversion of free antibody to HRP conjugate.

Complete conversion of antibody to conjugate greatly simplifies downstream purification. Purification consists of selectively binding the conjugate to a novel Q spin filter membrane that allows excess HRP to flow through unbound. The spin filter provides high purity without sacrificing conjugate yield. Conjugates made with All‐in‐One kit are compatible with all downstream applications such as western blots, ELISAs, or IHC. Each kit provides sufficient reagents to perform two (2) conjugation reactions; each yielding between 50‐70 μg of high purity HRP‐antibody conjugate.

B. All‐in‐One Technology

Conjugation Chemistry

The SoluLINK bioconjugation technology is based on the use of two complementary heterobifunctional linkers; S‐HyNic and Sulfo‐S‐4FB (Figure 1). S‐HyNic (Succinimidyl‐6‐hydrazino‐nicotinamide) is first used to modify and incorporate protected aromatic hydrazines (HyNic groups) into the antibody via acylation of lysine residues. In a similar fashion a second linker, Sulfo‐S‐4FB (Sulfo‐N‐succinimidyl‐4‐formylbenzamide) is used to provide a pre‐activated high activity HRP called 4FB‐HRP (included). Incubation of HyNic‐modified antibody with pre‐activated 4FB‐HRP in the presence of aniline catalyst leads to rapid and efficient conversion of the antibody to conjugate through formation of stable bis‐aryl hydrazone bonds (Figure 2).

Figure 1. Structure of S‐HyNic and Sulfo‐S‐4FB linkers used for conjugating HRP to antibody.

Figure 2. Aniline catalyzed conjugation of HyNic‐modified antibody with pre‐activated 4FB‐HRP.

Conjugate Purification

The efficiency of aniline‐catalyzed hydrazone bond formation greatly simplifies conjugate purification. Aniline’s ability to increase both the rate and efficiency of conjugate formation under mild reaction conditions leads to quantitative conversion of free antibody to conjugate. The complete absence of free antibody at the end of the catalyzed reaction leaves only two components; excess HRP and conjugate. After conjugation, a novel Q spin filter is used to selectively bind the conjugate based on known biophysical properties of IgG (4, 5) while allowing free HRP to flow through. Purified conjugate can then be eluted from the filter membrane free of both residual antibody and HRP in high yield (50‐70 μg).

Figure 3. Q spin filter purification of HRP‐IgG conjugate.

Figure 4. HRP‐Antibody All‐in‐One conjugation process.

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.

Description

Specifications

What’s in the box?

Materials suitable to perform 10 end labeling reactions of up to 0.6 nmols of 5′ ends (e.g. about 5 μg of a 25 base oligo) per reaction. This includes:

• T4 polynucleotide kinase
• 10x reaction buffer
• ATPγS
• Precipitant
• Alkaline Phosphatase

*Note: Thiol-reactive labeling reagent is not included in the kit and must be purchased separately. Labels suitable for this kit include:

• Biotin (Long Arm) Maleimide
• DNP Maleimide
• Fluorescein (Long Arm) Maleimide
• Fucose Maleimide
• Texas Red^® Maleimide

Note regarding shipping: This item is shipped on wet ice and therefore requires additional shipping charges.

Documents

• User Guide
• Safety Data Sheet
• Download CoA
• Datasheet

Technical Information

End labeling is a favored method for applications in which the presence of an internal label might interfere with hybridization or sequence specific protein binding.

5′ or 3′ EndTag labeled nucleic acids can be used for applications such as DNA hybridization, PCR, in situ hybridization, the binding of capture probes to affinity matrices, or electrophoretic mobility shift assays (EMSA). Short oligonucleotides are labeled more efficiently with these systems than with other methods. In addition, end labeling of oligonucleotides is an economical alternative to having labels inserted during synthesis.

Both the 5′ EndTag and 3′ EndTag Nucleic Acid Labeling Systems enable the covalent attachment of a variety of fluorescent dyes, haptens, or affinity tags to the respective ends of a nucleic acid using thiol-specific chemistry. Labels containing thiol-reactive groups (maleimides, iodoacetamides, etc.) can easily be incorporated.

Labeling time is about 1 hour with very little hands-on time.

5′ EndTag Labeling Kit

The 5′ EndTag System labels 5′ ends of DNA, RNA, or unmodified oligonucleotides. This kit is ideal for labeling PCR primers because a label is attached only to the 5′ end, leaving the 3′ end available for polymerization.

Labeling with the 5′ EndTag Kit is achieved in two steps:

1. T4 polynucleotide kinase transfers a thiophosphate from ATPγS to the 5′ -OH group of the nucleic acid. (5′ phosphorylated ends are converted to 5′ -OH groups with the included alkaline phosphatase).
2. Thiolated sample is coupled to a thiol-reactive label. The 5′ EndTag Kit is designed to perform 10 labeling reactions of up to 0.6 nmols of 5′ ends (e.g. about 5 μg of a 25 base oligo) per reaction. Thiol-reactive label is not included in the kit and should be selected separately.

Figure 1: EtBr-stained gel of a 1002 bp PCR product amplified using:
Ln 1) Unlabeled primers
Ln 2) EndTag biotinylated forward primer and unlabeled reverse primer
Ln 3) Unlabeled forward primer and EndTag biotinylated reverse primer
Ln 4) EndTag biotinylated forward and reverse primers.

Figure 2: Transmission electron micrographs of molecules of influenza A viral ribonucleoprotein particles (vRNPs) labeled at the 5′ end of the vRNA with biotin using 5′ EndTag Kit, and further labeled with streptavidin gold. vRNPs from influenza A were purified according to Kemler et al., on a glycerol gradient with modifications as described in Wu et al. Courtesy of Drs. Winco WH Wu and Nelly Panté, University of British Columbia, Vancouver BC, Canada.

Figure 3: Nuclear import assay in digitonin-permeabilized HeLa cells of biotinylated vRNPs. vRNPs were labeled first at the 5′ end of the vRNA with biotin using the 5′ EndTag Kit and then with Vector Fluorescein Streptavidin. This allowed for direct fluorescence visualization of the vRNPs on a confocal fluorescence microscope. Nuclear import assays were carried out as described in Wu et al. The negative control consists of vRNPs added to the cells in the absence of energy and exogenous cytosol. In the presence of energy and cytosol, the fluorescein-labeled vRNPs successfully enter the nucleus, with a high degree of nucleolar staining. Courtesy of Drs. Winco WH Wu and Nelly Panté, University of British Columbia, Vancouver BC, Canada.

Figure 4: Fluorescence in situ hybridization of human chromosomes using 5′ EndTag Fluorescein-labeled pUC1.77 detected directly and mounted in Vectashield Mounting Media with PI.

Figure 4: Coherent anti-Stokes Ramen scattering (CARS) and two-photon fluorescence (TPF) imaging of fluorescein-labeled HCV RNA inside Huh-7 cells. HCV RNA in-vitro transcripts were labeled with a fluorescein-maleimide label using the 5′ EndTag Nucleic Acid Labeling System. Huh-7 were transfected with 3µg of 5′-fluorescein-labeled HCV RNA and imaged at 4 h post-transfection. CARS and TPF of Mock transfected Huh-7 cells. Figure courtesy of Jennifer Haley, Sylvie Belanger, Adrian Pegoraro, Albert Stolow, and John P. Pezacki, The Steacie Institute for Molecular Sciences, The National Research Council of Canada. Unpublished data.

Figure 5: Coherent anti-Stokes Ramen scattering (CARS) and two-photon fluorescence (TPF) imaging of fluorescein-labeled HCV RNA inside Huh-7 cells. HCV RNA in-vitro transcripts were labeled with a fluorescein-maleimide label using the 5′ EndTag Nucleic Acid Labeling System. Huh-7 were transfected with 3µg of 5′-fluorescein-labeled HCV RNA and imaged at 4 h post-transfection. Here, CARS and TPF of Huh-7 cells transfected with fluorescein-labeled HCV RNA. Figure courtesy of Jennifer Haley, Sylvie Belanger, Adrian Pegoraro, Albert Stolow, and John P. Pezacki, The Steacie Institute for Molecular Sciences, The National Research Council of Canada. Unpublished data.

Figure 7: Fluorescence in situ hybridization of human chromosomes using 5′ EndTag Texas Red-labeled pHuR 98 detected directly and mounted in Vectashield Mounting Media with DAPI.

Description

3′ end labeling is the preferred method for oligonucleotide labeling if the terminal phosphate at the 5′ end must be preserved.

The 3′ EndTag System enables simple and uniform labeling of blunt, overhanging, or recessed 3′ ends of DNA. Using this system, a variety of fluorescent dyes, haptens, or affinity tags can be covalently attached to the 3′ end using thiol-specific chemistry. Additionally, labels containing thiol-reactive groups (maleimides, iodoacetamides, etc.) can easily be incorporated.

Specifications

Kit Components

What’s in the box?

The 3′ EndTag Labeling Kit is designed to perform 20 labeling reactions of up to 0.5 nmols of 3′ ends (e.g. about 4.2 μg of a 25 base oligonucleotide) per reaction. This kit includes:

• Terminal Transferase (TdT)
• SH-GTP
• 10x TdT buffer
• Precipitant

*Note: This kit requires Thiol-reactive labeling reagent, which is not included in the kit and must be purchased separately. Labels suitable for this kit include:

• Biotin (Long Arm) Maleimide
• DNP Maleimide
• Fluorescein (Long Arm) Maleimide
• Fucose Maleimide
• Texas Red^® Maleimide

Note regarding shipping: This item is shipped on wet ice and therefore requires additional shipping charges.

Documents

• Safety Data Sheet
• User Guide
• Download CoA
• Datasheet

Technical Information