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 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

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

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

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.

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.

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 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

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

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

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.

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 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.