Methods Digest, Vol 30, Issue 20

Pow Joshi via methods%40net.bio.net (by pow.joshi from gmail.com)
Sun Dec 2 16:20:14 EST 2007


On 30/11/2007, doaa zineldeen <zineldeen from gmail.com> wrote:
>
> hello
> I am having problem in detection of my protein which is a 72 KD kinase by
> western blotting
> I am using a commercial one , it could detect my kinase by IF howevere on
> western blotting many faint bands are seen and high background
> I tried different blocking and different dilutions of the primary and
> secondary and increasedwashing time but no clear band
> anyone can help




would you please let us know whether you see greater number of bands lower
to 72kDa or higher? Also, if it is a comercial Ab, they do have a data sheet
that gives you a complete characterization of the Ab...you could check and
see if they do use it for westerns, and what their recommendations
are....(if you havent already done so) ... further, is your kinase also
autophosphorylated?  in which case, would you be using phospho specific
epitope?

best,
Pow



thank you
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