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silver stain of immunoprecipitate


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#1 jasmina

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Posted 18 May 2010 - 01:59 PM

If we see on silver stain gel, many bands in addition to the band of protein of interest,
and by western blot, only the band of protein of interest,
does it mean that crossling IgG to beads by DMP didnt work??? or how can we explain these bands on silver !!

#2 Inmost sun

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Posted 19 May 2010 - 03:33 AM

If we see on silver stain gel, many bands in addition to the band of protein of interest,
and by western blot, only the band of protein of interest,
does it mean that crossling IgG to beads by DMP didnt work??? or how can we explain these bands on silver !!


immunoprecipitation is a method of enrichment which will not result in a pure fraction of a single protein if you start with a crude protein mixture; however, the result from the Western blot seems to show the specific enrichment; the impurities which are to be seen after silver staining seem to result from unspecific bindings to beads and IgG

#3 jasmina

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Posted 20 May 2010 - 02:55 AM

thanks,
in fact, i checked again the film of western blot, and there is also a faint band of the antibody!! , not as strong as the band of my protein.
should i check th crosslinking step?
i read that it's better to check the pH of DMP before and after incubation, and if its less than 8, it could reduce its efficiency!!
what do u think, should i measure ph of DMP?
thanks




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