Western blots - fuzzy below 100 kDa - (Jan/17/2006 )
A tech in our lab is trying to troubleshoot a western problem. On 10% SDS-PAGE gel of sperm extracts the dye front appears to be running higher than expected. Normally the dye front can be run off the gel and proteins as small as 10 kDa are still present at the bottom of the gel. Now the gel has to be stopped before the dye front has run out of the gel or proteins smaller than 25 kDa have run off the bottom of the gel. During the SDS-PAGE run, the dye front initially stacks appropriately but then appears to become unfocused during the run. Midway down the resolving gel a bright red line (which is very focused in contrast to the dye front) appears and it runs ahead of the dye front by a centimeter. After transfer of the protein by semi-dry blotting, there is good resolution of the samples at the top of the resolving gel (above 100 kDa), but the resolution is poor at the lower end of the gel with a lot of fuzziness below 100 kDa. Any suggestions as to what might be happening?
What kind of conditions are you using during your SDS-PAGE run? Are you using the large sized gels or mini-gels? I had a similar problem when running large sized SDS-PAGE gels and I attributed it to excessive heat due to the fact that my voltage during the run was too high, although I haven't yet re-run the gels at a lower voltage to see if that would help.
I'd be interested in hearing other suggestions as to why this may be occuring!
this is only a theory, but if the red is separating out of your loading dye, then perhaps the dye is bad? that stuff generally lasts forever, but there are always exceptions. perhaps there has been some sort of pH change in the dye? has this phenomenon been seen with another batch of dye?
i've encountered similar problems using a tris-glycine (i assume that's what you're using) buffer system gel at 10% arylamide. there are a few things you can try...
- make new stocks. in particular, we found that when we used a new batch of TEMED, this problem was lessened, but not solved.
what you're seeing when the dye front and a bunch of smaller proteins pile up into one small region is (i believe) called the chloride front. the relative mobility of this front (and the proteins that co-migrate with it) is in large part determined by the % acrylamide in your gel. a 10% gel will have a Cl- front that runs at a higher apparent MW than a 12.5% or 15% gel.
that said, if you're looking for proteins that are smaller than 100kDa, you might consider using a higher percentage of acrylamide in your separating gel. alternatively, if you need to look at multiple proteins of varying MW, then you might consider running a 4-20% gradient gel.
another option is to use a different buffer system (tris-tricine gels work nicely for this, and can be made as easily as tris-glycine gels), or purchase a pre-cast gel that uses a MOPS or MES buffering system, like the NuPAGE gels from invitrogen.
if you'd like a protocol for running a tris-tricine gel, let me know and i'll email one to you. typically, these gels are used to separate proteins of low MW, but in fact, you can alter a few things and get very nice separation of both high and low MW proteins within the same gel.
i hope this helps,