No bands in non-reducing SDS-PAGE - (Dec/11/2007 )
Hi, this is my first time at this forum, and I would like to thank you all in advance. Maybe could any of you help me with a little problem, it's really getting me crazy. When I run my samples (an ethanol precipitated fraction from a crude extract) on reducing SDS-PAGE, everything is fine. But when I use the same samples in non-reducing conditions (the same sample buffer, but without DTT), I get something like a light blue cloud along the tracks, and nothing about bands!!! I've notice another extrange thing: as the front migrates, some "bubbles" appear where the non reduced samples are, while the rest of the gel, with reduced samples, continues ok. I've been searching for an explanation in the web, papers, books about SDS-PAGE... but I didn't find anything about that.
Does anyone know what is happening with my samples??????
You need to make sure the samples are soluble before electrophoresis. At least, spin the samples at top speed for 5 min to remove insoluble parts.
EtOH precipitation can cause trouble for protein solubility, esp the samples were dried before reconstitute them. Any other way that you can use to process the samples?
I vortex the samples to mix them well, and speed them before loading the gel... I'm not sure, but I think if my proteins were not soluble, they would appear as a dark spot on the top of the track, isn't it? But there is nothing at all, and when I do western blot, the "signal" appears spread along the tracks, more intense at the sides, I mean: if the track was the stairs, these would for the banisters ... About the other question, after precipitation I let the pellet to air dry for 10 minutes before adding PBS to reconstitute them, does it matter? I need ethanol precipitation because the protein I'm looking for seems to be in that fraction, so I must deal with it...
Sounded like the proteins were not soluble or in aggregation status. If you have to stick with ETOH ppt method, try and see if you dissolve it while it is still wet, and if you can, dialyse to remove ETHO. If you dont remove ETOH, the surface tension may cause your samples fly away right in front your eyes when you load them. Be sure to spin it and only take the supernatant.
Genehunter, THANK YOU VERY MUCH for your help. Maybe I must dialyse... sometimes I notice a slight smell like alcohol... but what is really annoying me is: why samples work when they are reduced!!! And... may it help if I add some urea to the sample buffer? Literature says this would solubilice the proteins...
SDS helps the solubility as a complex with the peptide backbone.
It could be a solubility problem, but I have the same problem when running the crude extract, which has no ethanol at all. I also use SDS in my sample buffer, it is the same I use when reducing my samples, just without DTT. Anyway, yesterday I've told to give an aliquot of my samples to someone in other lab, they do native page and thing they can solve the problem I'll let you know if they get nice result... and maybe then I could know where was the trick!
did you also boil the non-reduced sample?
Usually I do so, to get rid of aggregates.
Yes, Missele, I've tried it, as well as heating them for 15 min at 70ºC, or no heat at all... But my samples don't take care about any of these procedures, they just... don't show up!!
Usually I do so, to get rid of aggregates.
I am a new forum user. I have the same problem than you about non reducing conditions. The wild type protein form a dimer and I can get the band in both, reducing and non-reducing conditions (the monomer and the dimer, respectively). The problem is when I use extracts of the mutant protein (that in theory can't form dimers), I can see the monomer in reducing conditions but with the same extracts in non-reducing conditions I can't see any band. If mutant protein can't form dimers it must be as the monomer form! I also observe extrange migration patern (2 light blue bands along the tracks, but not continuous). wat kind of gel are you using? I'm usin comercial ones (Invitrogen, NuPage 10% Bis-Tris gel) with MOPS runing buffer and without DTT for non reducing conditions (and without b-mercaptoetanol in sample buffer).
I'm sorry I have not the solution, but maybe we can think something useful together, good luck!!