I applied 140, 120. and 100 volts and I have obseved a very random migration of protein sample. It may be due to presence of salts (imidazole, NaCl, TisCl) in the sample. Sometimes the sample gets heated. Any suggestions??
Dear Gaurav,
Many times, I have seen your question related to SDS-PAGE only. you can do one more thing, you make fresh & correctly all SDS PAGE chemicals and run @ 90-110 voltage. I have used voltage till 160 volt while working in IISc and worked fine. so better to make all things fresh and correctly and do the experiment again. I also faced same kind of problem and struggled a lot. so I changed all SDS PAGE chemicals and problem was rectified.
I hope it will work fine with you.
Good luck dear.
I know. my current problem is different. It's the presence of salts. I guess. I have prepared everything freshly. Normal samples run smoothly but the eluted samples have problems.
One possible alternative is to use drop dialysis. Desalt, say, 20 uL of each sample (it will only take 20 min. or so in a 0.025 um membrane) before mixing with sample buffer.
Dear Gaurav,
It will obviously depend on exactly what you are doing, but if salts are the problem, why not just omit all salts from your imidazole elution? In my hands, 200 - 300 mM imidazole does not affect migration in standard Laemmli buffer. I understand that if you are doing large scale production of a protein, and would like to run an aliquot on a gel for QC, this is not possible. In this case, I agree with Alejandro Martin above, that dialysis to desalt would work. Alternatively, recapture a small proportion of you protein on Ni:NTA beads, wash away the salts, and elute again with a suitable sample buffer containing imidazole.
Good luck!
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