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

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)

Documents

• User Guide
• Safety Data Sheet
• Bioconjugation White Paper
• B-9014 – ChromaLINK Digoxigenin Conjugation Calculator
• Troubleshooting Guide – Bioconjugation
• Download CoA
• Datasheet

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.

Description

The S-4FB heterobifunctional crosslinker is fundamental to the SoluLINK bioconjugation technology. Sulfo-S-4FB reacts with primary amines on proteins (through lysine amino acids) or amino-modified oligonucleotides or surfaces, introducing a 4FB linker that forms stable covalent conjugates with biomolecules possessing HyNic linkers. Sulfo-S-4FB contains a Sulfo-NHS ester, which makes this linker water soluble. No DMF or other organic solvents are necessary to dissolve the linker prior to use.

Specifications

Documents

• User Guide
• Safety Data Sheet
• Protein Modification Calculator
• Troubleshooting Guide – Bioconjugation
• 4FB-Oligonucleotide MSR Calculator
• 4FB-Protein MSR Calculator
• 4FB Oligonucleotide MSR Instructions
• 4FB Protein MSR Instructions
• Oligonucleotide Buffer Exchange and Desalting Protocol
• Protein Buffer Exchange and Desalting Protocol
• BCA Protein Assay Protocol
• Bradford Assay Protocol
• Download CoA
• Datasheet

Technical Information

Introduction to SoluLINK Bioconjugation Technology

This core technology is based on the formation of a covalent bond formed from an aromatic hydrazine and an aromatic aldehyde. S-HyNic 1 (succinimidyl 6-hydrazinonicotinate acetone hydrazone, SANH) is used to incorporate aromatic hydrazine linkers on biomolecules. S-HyNic is an amino-reactive reagent that directly converts amino groups on biomolecules and surfaces to HyNic groups. 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 biomolecule or surface directly leads to the formation of the conjugate (Figure 1). The conjugate bond is stable to 92℃ and pH 2.0- 10.0. The recommended pH for antibody conjugation is 6.0. Unlike thiol-based conjugation protocols where reducing reagents are required that that can compromise the activity of proteins by cleaving disulfide bonds, the HyNic-4FB conjugation couple leaves disulfide bonds intact. No oxidants, reductants or metals are required in the preparation of conjugate.

Figure 1: Schematic representation of the SoluLINK bioconjugation chemistry where an antibody is modified with S-HyNic to incorporate HyNic groups and a second protein is modified with S-4FB to incorporate 4FB groups. Conjugate is formed directly by simply mixing the HyNic-modified antibody with the 4FB-modified proteins.

Further enhancing the many advantages of the HyNic/4FB conjugation couple is the discovery by Dirksen et al. that showed that 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 TurboLINK Catalyst Buffer (aniline) to the reaction mixture converts >95% of the antibody to conjugate in ~2 hours using 1-2 mole equivalents of second protein.

The HyNic-4FB conjugation couple is chromophoric- the conjugate bond absorbs at 354 nm and has a molar extinction coefficient of 29,000 L/(mol*cm). This allows (1) real time spectrophotometric monitoring of a conjugate reaction, (2) ability to visualize the conjugate during chromatographic purification using a UV or photodiode array detector and (3) quantification of conjugation.

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 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

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

Documents

• Safety Data Sheet
• Download CoA
• Datasheet

Legal Information

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

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

Documents

• Safety Data Sheet
• Download CoA
• Datasheet

Legal Information

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

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

Documents

• Safety Data Sheet
• Download CoA
• Datasheet

Legal Information

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

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

Documents

• Safety Data Sheet
• Download CoA
• Datasheet

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 S-HyNic heterobifunctional crosslinker is fundamental to the SoluLINK bioconjugation technology. Sulfo-S-HyNic reacts with primary amines on proteins (through lysine amino acids) or amino-modified oligonucleotides or surfaces, introducing a HyNic linker that forms stable covalent conjugates with biomolecules possessing 4FB linkers. Sulfo-S-HyNic contains a Sulfo-NHS ester, which makes this linker water soluble. No DMF or other organic solvents are necessary to dissolve the linker prior to use.

Specifications

Documents

• HyNic Protein MSR Instructions
• User Guide
• HyNic-Protein MSR Calculator
• Safety Data Sheet
• Troubleshooting Guide – Bioconjugation
• Oligonucleotide Buffer Exchange and Desalting Protocol
• Protein Buffer Exchange and Desalting Protocol
• BCA Protein Assay Protocol
• Bradford Assay Protocol
• Download CoA
• Datasheet

Technical Information

Introduction to SoluLINK Bioconjugation Technology

This core technology is based on the formation of a stable aromatic bond that has a UV-traceable signal to indicate the real-time formation of the conjugate. This bond is a bis-aryl hydrazone formed from an aromatic hydrazine and an aromatic aldehyde. S-HyNic 1 (succinimidyl 6-hydrazinonicotinate acetone hydrazone, SANH) is used to incorporate aromatic hydrazine linkers on biomolecules. S-HyNic is an amino-reactive reagent that directly converts amino groups on biomolecules and surfaces to HyNic groups. 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 biomolecule or surface directly leads to the formation of the conjugate (Figure 1). The conjugate bond is stable to 92°C and pH 2.0- 10.0. The recommended pH for antibody conjugation is 6.0. Unlike thiol-based conjugation protocols where reducing reagents are required that that can compromise the activity of proteins by cleaving disulfide bonds, the HyNic-4FB conjugation couple leaves disulfide bonds intact. No oxidants, reductants or metals are required in the preparation of conjugate.

Figure 1: Schematic representation of SoluLINK Bioconjugation chemistry where an antibody is modified with S-HyNic to incorporate HyNic groups and a second protein is modified with S-4FB to incorporate 4FB groups. Conjugate is formed directly by simply mixing the HyNic-modified antibody with the 4FB-modified proteins.

Dirksen et al. show that 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 TurboLink Catalyst Buffer (aniline) to the reaction mixture converts >95% of the antibody to conjugate in ~2 hours using 1-2 mole equivalents of second protein.

The HyNic-4FB conjugation couple is chromophoric — the conjugate bond absorbs at 354 nm and has a molar extinction coefficient of 29,000 L/(mol*cm). This allows (1) real time spectrophotometric monitoring of a conjugate reaction, (2) ability to visualize the conjugate during chromatographic purification using a UV or photodiode array detector and (3) quantification of conjugation.

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

MHPH (Maleimide HyNic) converts thiol groups on proteins and surfaces to HyNic groups, which is one of the fundamental linkers to SoluLINK bioconjugation technology. HyNic reacts with 4FB groups on other biomolecules or surfaces to create stable aromatic hydrazone bonds which are chromophoric and may be easily quantified spectrophotometrically.

Specifications

Documents

• HyNic Protein MSR Instructions
• User Guide
• HyNic-Protein MSR Calculator
• Safety Data Sheet
• Troubleshooting Guide – Bioconjugation
• Oligonucleotide Buffer Exchange and Desalting Protocol
• Protein Buffer Exchange and Desalting Protocol
• BCA Protein Assay Protocol
• Bradford Assay Protocol
• Download CoA
• Datasheet

Technical Information

MHPH (catalog number: S-1009) is a maleimide that converts thiols on biomolecules and surfaces to HyNic linker molecules for conjugation to 4FB-modified biomolecules.

Introduction to SoluLINK Bioconjugation Technology

This core technology is based on the formation of a stable aromatic bond formed from an aromatic hydrazine and an aromatic aldehyde. S-HyNic 1 (succinimidyl 6-
hydrazinonicotinate acetone hydrazone) is used to incorporate aromatic hydrazine linkers on biomolecules. S-HyNic is an amino-reactive reagent that directly converts amino groups on biomolecules and surfaces to HyNic groups. 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 biomolecule or surface directly leads to the formation of the conjugate (Figure 1). The conjugate bond is stable to 92°C and pH 2.0-10.0. The recommended pH for antibody conjugation is 6.0. Unlike thiol-based conjugation protocols where reducing reagents are required that that can compromise the activity of proteins by cleaving disulfide bonds, the HyNic-4FB conjugation couple leaves disulfide bonds intact. No oxidants, reductants or metals are required in the preparation of conjugate.

Figure 1: Schematic representation of the SoluLINK bioconjugation chemistry where an antibody is modified with S-HyNic to incorporate HyNic groups and a second protein is modified with S-4FB to incorporate 4FB groups. Conjugate is formed directly by simply mixing the HyNic-modified antibody with the 4FB-modified proteins.

Further enhancing the many advantages of the HyNic/4FB conjugation couple is the discovery by Dirksen et al. that showed that 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 TurboLink Catalyst Buffer (aniline) to the reaction mixture converts >95% of the antibody to conjugate in ~2 hours using 1-2 mole equivalents of second protein.

The HyNic-4FB conjugation couple is chromophoric – the conjugate bond absorbs at 354 nm and has a molar extinction coefficient of 29,000 L/(mol*cm). This allows (1) real time spectrophotometric monitoring of a conjugate reaction, (2) ability to visualize the conjugate during chromatographic purification using a UV or photodiode array detector and (3) quantification of conjugation.

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.