Western Blot Smearing?

d.white at genesis.co.nz d.white at genesis.co.nz
Tue Aug 27 18:16:46 EST 2002


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.

Thanks,

Damian.







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