SDS-PAGE running strangely - (Jun/17/2004 )
you should run the gels 60V until they all lined up between the stacking and seperating gels and then you can change the voltage to MAXIMUM 120V!! when you go to 200V your gel will finish faster but the the band look like crap
Hi, sometimes i think it can be the marker problem. I also had the problem that the protein band is larger than the expected size. But when i run with other markers, the same protein seems to be fine although its not the same sample as the one i run with the problematic marker. Perhaps you could try dual colour marker?
it could be that your gel is of low acrylamide percentage so your low mol. wt. proteins are running off.
there are many easons for that and the reasons are listed well by many helpers. But I want to say something. The markers are not readily used I mean some of them should be denatuarated at 95 celcius about 5 minutes however if you pass the time longer ( I once experinced) the larger proteins denaturadted so badly that I missed 116,2 Kda protein . generally the resons for all the meantioned above are related with low molecular weight protein marker however mine case s just the opposite
i had the same problem with you.
The problem with us was ' Running buffer pH'
Checked the running buffer pH.
Here the problem: I'm doing SDS PAge at 15 % acrylamide and before to put my film (autoradiography) I have to dry my gel. For a 1,5 mm gel thin, it takes 4 hours to dry at 80°c under vacuum. So after the electrophoresis I put my gel 30 min in a fixation bath (H2O, acetic acid, methanol), then, twice in water for 15 min and in glycerol 2% over night. In dry it in the morning.
Is there someone who can know if it's OK because somotimes my gel exploses, or I have some diffuse bands.
Thanks for all !
Your protein is glycosylated.
Is that the protein surface charge that involve in the migration of protein on SDS PAGE?
Thank you in advance.
On SDS PAGE, the protein migrate depending on its MW not the charge because the SDS mask the charge. try to boil the protien in sample buffer, when I say boiling means, boiling at 100C and for at least 5 or 10 mins.
I have a question folks. Some people use in regular lammeli buffer *M urea and DTT , When I boil the sample with such buffe and run on gel I do get multiple bands possibly of protein degradtion. Is it good idea to boild sample with such sample bufer? The seprating and satcking gels are of regular compostion. Pleae advise?
This may be due to carbamylation of your protein(s) resulting from the thermally-induced breakdown of urea into ammonium cyanate that then (as isocyanic acid) results in the carbamylation of amino-groups, adding approximately 40 Daltons per single modification event. What happens if you don't boil the samples?
According to the molecular mass was overestimated or lessestimated on SDS-PAGE maybe that the special amino acid composition.
High content of hydrophobic residues possibly results in poor binding of SDS, and then run slower;
low content of hydrophobic residues possibly results in strong binding of SDS, and then run faster;
Following is the weblinker for hydrophobic content assay: