Samuel C. Blackman
blackman at tigger.uic.edu
Sat Jul 28 19:07:09 EST 2001
You may wanto pre-conjugate your primary antibody with your secondary
antibody. This may help reduce some of the non-specific labeling that
you are having (especially if your protein of interest is in the 40-45kD
range. When you IP and then boil your beads you are going to denature
your primary antibody and then generate immunoglobulin HC and LC
fragments that can be picked up by your secondary antibody. If your
protein of interest is a particularly high- or low-MW protein, then
your problem may originate from a different source.
p.s.: There's a BioTechniques paper that describes this technique well.
You can search their on-line journal for 'immunoprecipitation' and you'll
be sure to find it.
In article <9jjkgr$ssa$1 at niobium.hgmp.mrc.ac.uk>,
nsampson at hgmp.mrc.ac.uk (Natalie Sampson) wrote:
> I am trying (without success) to pull down a transiently expressed c-myc
> epitope tagged protein. From immunocytochemistry labelling, my protein is a
> soluble nuclear factor. From the precipitation experiments I have done so
> far, I am getting several background (unwanted proteins) and not mine. It is
> definitely present in the transfected cells (western blot analysis on total
> cell lysates). Does anyone have any suggestions. I have tried numerous
> washing buffers (RIPA, high salt etc) and am fast running o
> ut of ideas. Can anyone help?
> Thank you
> nsampson at hgmp.mrc.ac.uk
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