All small peptides/haptens must be coupled to a carrier protein (KLH, BSA, Ovalbumin, etc) in order to elicit high titer antibodies. Generally, peptides can be coupled to other proteins by utilizing:  

1.   A free NH2 or COOH (recommended for peptide when Cysteine cannot be added to the peptides).
2.   A free Cysteine group (available as part of the sequence or added at the N or c-terminus)-preferred method of coupling peptides.  

Chemical conjugation using Cysteine offers a single point attachment provided there is just one Cys in the sequence (added or part of the native sequence).  It is preferable to add Cys at the NH2 terminus if the peptide is internal or it represents the very C-terminus. This will keep the COOH free (non-conjugated) as it exists in native protein.  For peptides representing the very NH2-terminal sequences, Cys should be added at the C-terminus of the peptide. For internal peptides, Cys can be added at either end but it is easier to synthesize peptides containing a NH2-terminal Cysteine.  

We use MBS (m-Maleimidobenzoyl-N-hydroxysuccinimide Ester) method for peptides containing Cysteine. Glutraldehyde method is used coupling via the free amino group and EDAC (1-Ethyl-3-(3′-dimethylaminopropyl)carbodiimide) for free COOH.     

BSA and KLH proteins are pre-activated with MBS and supplied in powder form.  All other necessary buffers are provided for coupling 2-4 mg of the peptide in about 3-4 hrs at room temp.  The conjugated peptides are ready for immunization.  Each kit contains 2x 2 mg of the pre-activated proteins (BSA or KLH) and other buffers to couple 2 different peptides.  Easy to follow directions are provided with the kit.  

Cysteine can also be used to couple peptides to Sepharose for affinity purification of antibodies. Amino or COOH-conjugation chemistries should be avoided as most peptides contains several NH2 and COOH groups available in a given peptide sequence resulting into multi-point attachment and peptide distortion.  

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