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White speckles on western blots


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3 replies to this topic

#1 seanspotatobusiness

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Posted 10 June 2009 - 07:57 AM

Sometimes I get white speckles on my blots, as though the blotting transfer was interfered-with somehow. The same is apparent regardless of whether they are stained with Coomassie blue or antibody-HRP/AEC stain.

Both these images are pretty small (the best I could do with our scanner) but if you zoom in (Ctrl + Scroll wheel on mouse) you can see the problem.

http://www.petaimg.c...361crapblot.tif
http://www.petaimg.c...89crapblot2.tif

My supervisor has less experience than I do, and the woman-of-experience in the lab across the hall has no suggestions. Any clue?

#2 madrius1

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Posted 10 June 2009 - 10:42 AM

Any chances that the membrane and the gel didn't make contact all the way through during the transfert?

Or is it possible that the pouch you incubate your membrane in with the antibody got stuck onto the membrane at that specific spot, therefore preventing the ab to bind?

#3 Dominic

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Posted 11 June 2009 - 03:08 AM

sounds like bubbles tween gel and paper - try using a piece of plastic pipette as a rolling pin to ensure full contact (do it over the sponges)

d

#4 mastermi

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Posted 11 June 2009 - 05:50 AM

Yes, most probably it's bubbles between gel and membrane during blot.
Or did you accidentally grab on the membrane ? I have also seen speckles like that after a student grabbed on the membrane...




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