R-PE Antibody Conjugation Kit

P-9002
SKU Unit Size Price Qty
P-9002-002 1 Kit
$528.00

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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

More Information
Conjugation Targets Antibody
Label/Modifier Type R-PE
Reactivity Amine
Recommended Storage 4°C
Applications Antibody Labeling, Antisense/RNAi, Aptamers, Immunotherapeutics, In Situ Proximity Ligation, In vitro Diagnostic (IVD), Laboratory Developed Tests (LDT), Analyte Specific Reagents (ASR)

Kit Components

  • S-HyNic (1.0 mg vial)
  • 4FB-R-PE (1.5 mg)
  • 10X Modification Buffer (1.5 mL vial)
  • 10X Conjugation Buffer (1.5 mL vial)
  • 100 mM Aniline Buffer (1 mL vial)
  • 0.5 mL Zeba (4)
  • DMF (anhydrous) (1 mL vial)
  • 2-Sulfobenzaldehyde (100 mg)
  • 10X MES Buffer (1.5 mL vial)
  • Collection tubes (12)

Documents

Technical Information

R-PE-Antibody Conjugation Kit Using SoluLINK’s HydraLINK Technology

Introduction: SoluLINK has engineered its HydraLINK conjugation technology to more easily and efficiently prepare R-PE-antibody conjugates compared to past maleimido/thiol-based protocols. The kit includes all the reagents and buffers needed to perform the two step conjugation.

Advantages: SoluLINK’s R-PE-antibody bioconjugation technology is superior to the maleimido/thiol-based method as it is:

More efficient: 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.

More easily purified: 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. In many applications purification is not necessary.

Controllable: The level of polymerization, and therefore the brightness of the conjugate, can be controlled by adjusting the level of HyNic modification on the antibody. In flow cytometry applications, heterodimer product is preferred and preparation of this construct can be easily optimized using this technology.

and furthermore

An intact antibody is incorporated: Other conjugation methods expose thiols on antibodies by DTT reduction of disulfide bonds, which cleaves the antibody into a variety of species. SoluLINK’s technology, however, gently incorporates HyNic moieties on the intact antibody.

Introduction to SoluLINK Bioconjugation Technology

SoluLINK’s core 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 1 (succinimidyl 6-hydrazinonicotinate acetone hydrazone, SANH) is used to incorporate aromatic hydrazine linkers on biomolecules. S-HyNic is an amino-reactive modification 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 R-PE leads to the formation of the conjugate via a bis-arylhydrazone bond. The bis-arylhydrazone bond is stable to 92OC and pH 2.0-10.0. The recommended pH for antibody conjugation is 6.0.

Figure 1: Scheme presenting the two-step protocol to prepare R-PE-antibody conjugates using SoluLINK’s HydraLINK bioconjugation technology. Step 1 is modification of Antibody with S-HyNic to prepare HyNic- Antibody. Step 2 is the conjugation of HyNic-Antibody to 4FB-R-PE simply by mixing in 100 mM phosphate, 150 mM NaCl, 10 mM aniline, pH 6.0.

The many advantages of the HyNic-4FB conjugation couple include that:

1)The reaction is high yielding. Routinely yields of conjugate are 40-60% based on starting protein.

2)The reaction is efficient: Dirksen et al. discovered that aniline catalyzes the formation of 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.

3)The conjugate bond is stable: The bis-arylhydrazone conjugate bond is stable to 92OC and pH 2.0-10.0

4)The reaction conditions are extremely mild and do not cause any 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.

5)The conjugation is traceable spectrophotometrically. The HyNic-
4FB conjugate bond is chromophoric- it absorbs at 354 nm and has a molar extinction coefficient of 29000. 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.

The Keys to Success

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

  1. Antibody buffer exchange: Prior to modification, the starting Antibody must be completely exchanged into Modification Buffer, pH 8.0.

  2. Minimum level of HyNic-modification: The HyNic-antibody molar substitution ratio (MSR) must be >3.5 as determined by a colorimetric assay.

  3. The final concentration of the HyNic-antibody in the conjugation reaction must be >1.5 mg/mL.

 

Intellectual Property

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.

TriLink does not warrant that the use or sale of the products delivered hereunder will not infringe the claims of any United States or other patents or patents pending covering the use of the product alone or in combination with other products or in the operation of any process. All and any use of TriLink product is the purchaser's sole responsibility.