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maddy27 05-23-2012 07:51 AM

problem with transfer and western blotting of BN-PAGE
 
Hello there,
Im trying to do BN-PAGE followed by western of the same on to PVDF. In my first experiment I ran only native markers(10% resolving gel) and stained the gel with coomassie and I can see the various markers clearly. Now when Im running my samples followed by western transfer on PVDF, I dont see clear bands with Ponceau on the PVDF. Anyway I proceeded with the blot and I see hazy background on my x-ray sheet apart from some bands.
I want to know why this is happening. After transfer I wash the membrane with methanol to remove G250 coming from BN-gel, later give washes for 15 minutes with TBST before adding 5% blotto. Please let me know what can be the reason for the hazy background.


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