protein binding to niotrocellulose

Jarrat Jordan jarjor at PEACHNET.CAMPUS.MCI.NET
Sat Dec 13 13:53:07 EST 1997


I conjugated a 20 mer synthetic peptide to KLH and had the antigen used
to immunize mice. On receiving my test bleed I tested the serum by
pipetting 1 microgram of pure KLH and 1/10 and 1 microgram of the
purified 20-mer to a nitrocellulose membrane. After developing the blot
with HRP, only antibodies against the KLH appeared to be present. My
question is: Is a 20 residue synthetic peptide large enough to withstand
the washing and processing of a blot, could this 20 mer have simply
washed off leaving the larger KLH? If so what procedure should I use to
confirm the presence of anti hapten antibodies? Does anyone know of any
literature pertaining to this? The KLH and the synthesized peptides
where pipetted in solution on the membrane and allowed to air dry. After
this the membrane was blocked with calf serum and processed like a
typical Western.
Please help me with this, thank you
Jarrat Jordan UGA microbiology




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