strange and branch like bands on SDS-PAGE - troubleshooting (Jun/30/2011 )

Hi, i have run many SDS-PAGES during working on my projects but recently I faced a problem.to check the purity of a recombinant peptide I am loading that after purification with Ni-NTA but after staing the gel all bands are duiffused and in so strange shape. There are no sharp bands and just bands like branches!I tried to prepare all buffers freshly and also I run at 80V for the first layer and 120 for the second but stil I couldnot overcome. could you please help me in this case?

pEXP23 on Thu Jun 30 14:15:10 2011 said:

Thank you so much for your kind welcome:) Please find my gel image. I should mention that i had stored my samples at 8 degree for about 4 days. and after that I found out that it has some presipitate. Before this the gel has two sharp bands.

HOw big is your peptide, and what percent gel did you run?

Protein samples should never be stored at fridge temperatures unless they are in a lyophilised state. Always store at a minimum of -20 deg C.

The peptide is about 13.5KD and the percentage of gel is 17%.So maybe the protein is degraded

IMHO, it looks like not well polymerized GEL

I would suggest:
Change to newly prepared /purchased APS. Use it fresh.
Use a multichannel pipette to load your acrylamide in between the glass when you make your gel. DO not load in one-side.
Use a lower voltage to avoid overheating, better you run your PAGE inside ice-box (I always do that).

Thank you for your suggestions. But in fact I did all of these using fresh buffers and APS. And also one time I put ice outside the tank and also reduced the voltage to 100v:( anyway I will try it again.

I dont know if it still going to help but I had a similar problem, and the reason was that the ph of the samples I was loading was too acidic, so after neutralizing it a little bit more, everything was perfect