protein binding to niotrocellulose

Rod Macfarlan rmacfarl at csl.com.au
Sun Dec 14 18:57:21 EST 1997


On 13 Dec 1997 10:53:07 -0800, jarjor at PEACHNET.CAMPUS.MCI.NET (Jarrat
Jordan) wrote:

>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
>
 You have a number of approaches to detect the anti-peptide component
of the response:

1. Demonstrate, using a chemical test such as ninhydrin, that your
peptide has in fact bound to nitrocellulose. Do this before blocking
of course. Still won't eliminate the possibility that the antibody
can't bind due to steric factors.

2. Make a similar protein-peptide conjugate for analysis (by EIA or
dot blot). Use a different (non-crossreactive) protein, and take care
that you are not detecting anti-linker antibodies. This can be a
problem with commonly used maleimido bifunctional reagents, in which
case the best approach is to conjugate using a different chemical
reagent. As to the protein, we use casein which is also a component of
our blocking solution thereby reducing background to nil.

3. Use any of a number of approaches to bind the peptide to the solid
phase in a defined site-specific fashion - with a spacer if possible,
and in the same orientation as the conjugate used for immunization. We
routinely biotinylate the peptide at the N-terminal and use
streptavidin coated plates, however there are dozens of possible
alternatives. Two that spring to mind are chemical coupling to
amine-derivatized solid phases, or other affinity methods such as
chelation of his6-tagged peptides by NTA-plates from Pierce.



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