Re:antibody testing

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From:Joyce Kotzuk <> (by way of histonet)
To:histonet <>
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Just a few more detailed thoughts on antibody testing for the first time:
      If I really have no idea of which concentration/dilution the antibody
in question is going to work at, I do a wide range of dilutions on the
first run (ie. 1:50/ 1:500/ 1:2,000.)  This usually gives me a good idea of
where to go from there, with the advantage of saving potentially expensive
antibody if it turns out that the antibody works well at a high dilution
(low concentration). Often, the first "procedure variable" that I test is
whether or not to microwave the section in antigen retrieval buffer. From
there I test other procedure manipulations such as blocking concentration,
NGS concentration in primary and secondary antibody, and other
pretreatments such as Triton-X, formic acid, avidin/biotin blocking etc.
Good luck.

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