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Questions about Blotting of Low Molecular Weight Peptide


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#1 hlb10

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Posted 01 December 2010 - 07:39 AM

Hi fellow scientists,

I am working on detecting a low molecular weight peptide (12.5kd) in tissue lysate.

Some basic background...antibodies were produced by immunizing rabbits with a cross-linked peptide. These antibodies were found to have interesting properties in cell culture assays. In order to test these antibodies in an animal model, an investigator asked to see that these antibodies had reactivity against animal tissue.

I started with a dot blot by depositing high concentrations of the cross-linked peptide directly on nitrocellulose membrane and found the antibody indeed had reactivity against tissue lysate but had no reactivity against high concentrations of the cross-linked peptide. Theoretically, this should be the expected reaction given the rabbits were immunized with cross-linked peptide and the peptide is now denatured, no? An adviser asks whether the cross-linked peptide was boiled in sample buffer...why would that matter?

An adviser also suggested I run the peptide through gel electrophoresis. Might I expect to see the cross-linked peptide after gel electrophoresis when there was no reactivity between the antibody and high concentrations of the protein directly deposited on the membrane?

Thanks.

#2 mdfenko

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Posted 01 December 2010 - 08:23 AM

boiling the peptide in sample buffer will denature it (as in sds-page).
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