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Blocking Buffer gone bad

Blocking buffer 5% milk western blot nitrocellulose membrane green

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#1 Iman Aaz

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Posted 10 December 2012 - 09:28 PM

Hey Guys,

I have noticed that over the past couple of days, blocking my nitrocellulose membranes in 5% TBSTM for more than two nights is causing them to go green, sometimes associated with a ver raw smell! This has never happened before, we used to block for a week sometimes with no issue whatsoever. Anyways, I washed several times with PBS until I could no longer smell that pungent smell and tried adding an old (frozen, reused) antibody I had that has been working perfectly fine, only when I developed I did not see anything. Any ideas as to what could be the source of the this milk going bad, and could it have deteriorated my proteins?

Thanks

#2 bob1

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Posted 11 December 2012 - 01:04 AM

Bacteria in the blocking buffer. Green ones are often pseudomonas aeruginosa, but could easily be E.coli. This will have degraded the proteins on the membrane and also degrade antibodies quite nicely.





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