I run 2D gel for my E.COLI sample in this condition ,7cm ipG strip (3-10 NL), IEF condition step 1)500 v for 1000 vh linear, step 2)3500 v for 10000 vh linear, step 3) 3500 v for 20000 vh rapid.....and rehydration buffer: 8 M urea, 2 M thiourea, 2 % CHAPS, 28 mM DTT, 1.3 % PHARMALYTES PH 3-10, trace bromophenol blue+ 25 :)microgeram protein. for second dimension i used 12% acrylamide gel and 150 v for 40 min..stain with coomasie blue.
Does any body know what is the problem of my protocol? why there is multipling in my spot?.. why there is alot of streak? what should i do?..I need your help..(I attached the picture of my gel)
what is the problem in my 2D gel?????
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