protein binding to niotrocellulose

David L. Haviland, PhD dhavilan at IMM2.IMM.UTH.TMC.EDU
Sun Dec 14 08:16:56 EST 1997


On 13 Dec 1997, 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

Jarrat:

I've done something similar in that I immunized mice with a 18-mer that
had been coupled to KLH as well.  What I did for screening and testing was
to couple the 18 mer again but this time I coupled it to OVA instead.
Using the KLH-18 mer in separte wells, aling side of the OVA-18mer, I used
an ELISA to determine the titer against KLH and against my peptide and I
could separate the response and asses to what extent the response was
directed to my peptide rather than the carrier.  It sounds to me as though
you need your peptide coupled to another carrier so you can see the
difference.

Hope this helps...
David

==========================
David L. Haviland, PhD
Assistant Professor, Immunology
University of Texas, HSC - Houston
Institute of Molecular Medicine
2121 W. Holcombe Blvd.
Houston, TX  77030
http://www.uth.tmc.edu/~dhavilan
713.500.2413-Voice
713.500.2424-FAX
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