First of you have a very reactive peptide that is going to covalently
couple at multiple sites to KLH resulting in very few exposed residue.
Assuming that you will need about 7 to 12 residues for a linear epitope, it
is unlikely that you have enough resiues exposed. One possiblity is to
derivatize using a amine reactive heterobifunctional linker, activate
peptide (only one free NH2) then couple to KLH or variation thereof. Next
is to add a Tyr to the C or N terminus of the peptide, depending as to what
region is antigenically important and use diazotization to couple to KLH, I
do not have the reference for the Tyr-coupling at hand but will post it
tommorrow. This will yield a brown muck, but it works!! Last, this peptide
is long enough to have internal disulfides, you might want to use 4-4'
dithiopyridine to block SH gropus, couple using Glutaraldehyde then deblock
the pyridyl groups with excess DTT, followed by dialysi into PBS or TBS.
more later, good luck, gautam
Gautam Sarath
N-226, Beadle Center
Protein Core Facility - Center for Biotechnology &
Department of Biochemistry
University of Nebraska-Lincoln
Lincoln, NE 68588-0664
Phone: 402-472-2928
FAX: 402-472-7842
http://www.biotech.unl.edu/Proteins/index.html