Western Blot Smearing?
H.Veldman-2 at neuro.azu.nl
Wed Aug 28 03:20:59 EST 2002
d.white at genesis.co.nz schreef:
> I'm currently having recurrent problems with western blot analysis, specifically with excessive smearing around the top of the blot, and frustration is setting in.
> I'm trying to detect expression of a specific protein from cell line lysates extracted with garden variet RIPA buffer, etc., using a rabbit polyclonal primary, and an anti-rabbit HRPO secondary.
> Coomassie blue staining of a SDS-PAGE gel reveals clear, consistent banding, yet upon western transfer and development, I get repeated smearing of bands from the top of the resolving gel to about the 60kDa mark, where our protein should theoretically run.
> The smearing takes the form of dots and vertical smears that appear to be independant of lane shape, as if there's a significant element of insolubility present in my lysates that causes fractionated solubility as they run in the presence of SDS. I've tried to spin my samples hard before loading to pellet any membrane-associated insoluble proteins, but without success.
> The lower half of the blots are very spotty also, and have been compared to a star chart . . . which doesn't do much for results . . .
> Faint secondary banding is evident in areas where this smearing and spotting doesn't overpower the blot, so things are behaving as they should on some level, but ultimately it's all too messy for conclusive results.
> If anyone recognises these symptoms and can offer some reprieve, I'd be greatly appreciative.
It is omnipresent and highly immunogenic, so:
At the one hand, when a rabbit is immunized, usually with the aid of an adjuvant, it will regularly develop high titers against human keratin too.
On the other hand, it is virtually impossible to avoid getting human keratin in your system during preparation, isolation, electrophoresis, blotting, etc.
The best way to get rid of this is using monoclonal antibodies or affinity-purified polyclonals. Secondary antibodies should be free of anti-human keratin cross-reactivity.
Laboratory for Experimental Neurology (NMZ)
University Medical Center Utrecht (AZU)
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