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Mysterious disappearing bands after blotting

Western blotting

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5 replies to this topic

#1 DrSnood

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Posted 07 August 2014 - 06:11 AM

Hi All,

 

Our lab has encountered a very strange problem recently with western blots. A few of us have successfully blotted for our protein of interest, only to produce blank films on the subsequent blot. For example, I recently blotted for my protein of interest, but wasn't satisfied with the way the blot turned out and didn't have time to deal with it that day. I put the membrane back in the same antibody prep overnight and tried to develop it the following day. My film was blank even after I left it down for over an hour. This also frequently occurs when blotting for loading proteins after successfully blotting for the protein of interesting. In most cases, the membrane has not been stripped, and in all cases, the marker is still visible on the membrane. We use PVDF (soaked in methanol and the transfer buffer prior to the transfer). This issue seems to occur regardless of the target protein (tags on overexpressed proteins, endogenous tricky proteins, loading controls). Is it possible for protein to diffuse off of PVDF? None of us can think of a reasonable explanation, and are pretty frustrated at this point. Any ideas are appreciated!



#2 bob1

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Posted 07 August 2014 - 01:09 PM

Its possible that either your antibody is decaying while you are doing the overnight incubation, or that you have a bacterial contamination of your block that is degrading the protein.



#3 DrSnood

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Posted 07 August 2014 - 01:40 PM

We've tried about 10 different antibodies and fresh block (new 1X TBS-T with 5% non-fat milk powder). What might make an antibody decay? And can non-fat dry milk powder go bad? Thanks!



#4 bob1

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Posted 09 August 2014 - 01:52 PM

Non-fat milk powder does degrade over time  -it oxidises and can easily get contaminated with bacteria that will happily grow once the powder is made into solution.



#5 DrSnood

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Posted 13 August 2014 - 12:06 PM

Ok, this is good information. Is there any way to salvage my membrane that was incubated in potentially bad milk solution? I really need to get my loading to work on this blot! Thanks!



#6 bob1

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Posted 13 August 2014 - 12:38 PM

You could try stripping the blot and then re-probing.







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