Ok here's a tough one,
We have been running western blot successfully here for quite a long time on pvdf membranes, it's been from time to time a little inconsistant, but we were always able to get good results after making a new blot, or reblotting. For about the last two months it has been very inconsistant (positive control there but nothing else, no positive control but samples with other bands...). Now here's the kicker, in the last two weeks we haven't been able to get any bands, even with our house keeping protein which has always worked extremely well. We have replaced almost all of our buffers with new ones. The marker appears after transfer, and there are bands on the Ponceau. Our ECL has been tested, and our hrp abs are all still good. Our ABs are stored in different locations, so they can't all possibly be bad, and our house keeper is in aliquots. We block with 5% milk, but always have, and we wash with TBST. Where is our problem, I would appreciate it if someone could help!
Thanks!
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#2
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Posted 15 June 2010 - 01:10 PM
Larry_Rollins, on Jun 15 2010, 04:15 PM, said:
Ok here's a tough one,
We have been running western blot successfully here for quite a long time on pvdf membranes, it's been from time to time a little inconsistant, but we were always able to get good results after making a new blot, or reblotting. For about the last two months it has been very inconsistant (positive control there but nothing else, no positive control but samples with other bands...). Now here's the kicker, in the last two weeks we haven't been able to get any bands, even with our house keeping protein which has always worked extremely well. We have replaced almost all of our buffers with new ones. The marker appears after transfer, and there are bands on the Ponceau. Our ECL has been tested, and our hrp abs are all still good. Our ABs are stored in different locations, so they can't all possibly be bad, and our house keeper is in aliquots. We block with 5% milk, but always have, and we wash with TBST. Where is our problem, I would appreciate it if someone could help!
Thanks!
We have been running western blot successfully here for quite a long time on pvdf membranes, it's been from time to time a little inconsistant, but we were always able to get good results after making a new blot, or reblotting. For about the last two months it has been very inconsistant (positive control there but nothing else, no positive control but samples with other bands...). Now here's the kicker, in the last two weeks we haven't been able to get any bands, even with our house keeping protein which has always worked extremely well. We have replaced almost all of our buffers with new ones. The marker appears after transfer, and there are bands on the Ponceau. Our ECL has been tested, and our hrp abs are all still good. Our ABs are stored in different locations, so they can't all possibly be bad, and our house keeper is in aliquots. We block with 5% milk, but always have, and we wash with TBST. Where is our problem, I would appreciate it if someone could help!
Thanks!
I remember a similar situation in our lab: A slight shift of pH (from PH7.4 to pH7.2) of non-carefully prepared PBS was our reason; you need definitely a positive control
#3
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Posted 14 October 2011 - 07:32 AM
I have this same exact problem, did you figure out what the cause was??
