Maleimide-Activated 6mg KLH Kit

Catalog #:  Pi-KLH-103

Peptides, by themselves, are too small to elicit an immune response or antibody production against the peptide.  Therefore, all peptides must be coupled to an immunogenic carrier molecule.  The traditional immunogenic carrier molecule is KLH (Keyhole Limpet Hemocyanin).  Several peptides can be conjugated to each KLH molecule. 

In the Maleimide-Activated KLH Conjugation Kit, the KLH is pre-activated using Sulfo-SMCC.  The activated KLH contains maleimide group that can react with the sulfhydryl group  (-SH) on the side chain of Cysteine between pH 6.5-7.5 to form stable thioether bonds. 

Kit  Components:
Maleimide-Activated-KLH (Blue Cap), contains 3 vials, 2mg/vial
Conjugation Buffer (Yellow Cap), contains 3 vials, 2mL/vials, 83mM sodium phosphate, 0.9M sodium chloride, pH 7.2

Storage:  Kit ships at ambient temperature. Upon receipt store kit at 4°C.

Different sizes and structure of peptides will affect conjugation efficiencies.  However, using a molar excess of peptide over maleimide groups on the KLH will ensure efficient conjugation.  Therefore, we recommend using equal mass amounts of peptide and KLH to achieve sufficient molar excess.  Maleimide reacts with the free sulfydryl group (-SH) on the Cysteine at pH 6.5-7.5.  This pH range will form a stable thioether bond.

1. Reconstitute one vial (2mg) of maleimide activated KLH (Blue Top Vial) with 500μL of conjugation buffer (Yellow Top Vial). Note: Do not heat or vortex the solution.  The solution may have a translucent bluish white appearance.
   
   
2. Dissolve the –SH containing peptide (2mg) with 500μL of conjugation buffer (Yellow Top Vial).  Note: If the peptide has poor solubility, add DMSO to the solution.  However, do not have more than 25% DMSO in the final conjugation solution. 
   
   
3. Add the peptide to the activated KLH and mix gently.
   
   
4. React for 3-4 hours at room temperature.
   
   
5. Perform dialysis on the peptide conjugate using PBS for 10-12 hours.  Change the PBS every 3 hours. You may also use a desalting column. 
   
   
6. Store the conjugated peptide at -20° or below in suitable size aliquots.   Note: Immunization amount for rabbits/chickens/mice will vary.  Check with your animal facility or staff veterinarian for proper amounts.

NOTES:
 
Free Sulfhydryl Group- The peptide must have a free sulfhydryl group (-SH) for proper conjugation.  Perform an Ellman’s Test to ensure the sulfhydryl group is available for conjugation.  If the sulfydryl groups are not available, the peptide must be reduced before conjugation. 

pH Matters-  At pH values greater than 7.5, reactivity toward primary amines and hydrolysis of the maleimide groups are possible.  Therefore, the pH must be between pH 6.5-7.5 to form stable thioether bonds.