Product name :Adar's Maleimide Activated OvA

Catalog Number :Cat.1026-10

Quantity :10 mg

Price :265 Eur

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Supplier :Adar Biotech

Introduction

Activated KLH, BSA and Ovalbumin (OVA), are commonly used as protein

carriers for haptens such as peptides in order to enable the immune response to

small molecules. In many cases KLH conjugate is used for the immunization, while

BSA or OVA conjugate is used in immunoassys of the resulting antibodies. OVA

conjugates are used in order to filter out the antibodies directed to the KLH.

However in some cases OVA or other proteins are preffered as carrier proteins

because of a variety of other reasons.

The Maleimide activated KLH and OVA produced by Adar Biotech are

preactivated with a heterobifunctional cross-linker (GMBS). These activated

proteins may be reacted with biomolecules that contain a free sulfhydryl groups, to

form a stable thioether bond.

 

Maleimide-Activated OVA characteristics.

Activation method: GMBS.

Binding capacity: ~2-4 mg of peptide (average MW of 1000-2500) per 2 mg OVA

Protein concentration: 4 mg/ml (0.5 ml)

Storage buffer: PBS pH 7.5

Transportation conditions: Ambient temperature up to 7 days.

Storage condition: -20°C.

 

A. Procedure for Peptide Conjugation

1. Dissolve the sulfhydryl-containing hapten in a volume of water (or suitable

buffer) equals to x0.4 to x1 times the volume of OVA. For example dissolve 2 mg

of peptide in 200-500 μl of buffer for addition to 2 mg of activated OVA in 500 μl.

 

Note: For haptens with limited solubility, DMSO may be used for solubilization. Use .30%

DMSO in the final conjugation solution or the carrier protein may irreversibly denature.

Alkaline pH values (above 8.5) may hydrolyze the maleimide group or generate side reactions

with amines. Haptens must contain cysteine or a sulfhydryl group in the reduced state in order to

react efficiently with the maleimide group.

 

2. Thaw the Maleimide Activated OVA at room temperature.

Note: Do not vortex or heat the activated OVA.

3. Mix the peptide with activated OVA adding peptide stepwise (1/4th of quantity

each time in 5 minutes intervals) and watching for possible appearance of turbidity

in solution. If slight turbidity forms – stop adding peptide to KLH solution.

Reaction is done for 2 hours at room temperature under occasional tapping of tube.

4. Peptide-conjugated OVA can be purified by gel filtration or dialysis to remove

unbound peptide

Note: If the immunogen is to be stored for more than a few days it is recommended to store

frozen at -20°C.

5. The coupling efficiency of conjugation can be determined by assaying the

content of free sulfhydryl groups in the unreacted peptide using DTNB reagent.

 

B. Storage

The activated-OVA should be stored frozen until use.

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