Presence of 'blobs' at the dye front in SDS-PAGE - (Oct/13/2010 )

Hi everyone,

Thanks for taking the time to look at my problem. I've been performing some subcellular fractionation experiments and creating total, nuclear, cytosolic and crude mitochondrial extracts using a Tris based buffer with 1% NP40 (50mM Tris-HCL pH 7.4, 150mM NaCl, 2mM EDTA, 2mM EGTA, 1% NP40, protease and phosphatase inhibitors). I've been trying to separate these extracts using a 12% SDS-PAGE gel (Tris-Glycine buffer system with a 4% Stacking gel), however a dye 'blob' keeps forming in each lane at the gel front. The 'blobs' become more apparent as the separation proceeds and a small purple band also forms infront of them over the course of the run. Furthermore, the 'blobs' are larger in the total and nuclear extracts compared to the cytosolic/mitochondrial extracts. I'm running the gel at constant voltage (100V).

Does anyone have an idea of what is happening and how i might be able to correct this problem?

Thanks again,

J

Sorry.. I forgot to say that the marker runs fine so i don't think it's an issue with the gel or the buffer..

Cheers,

J

a picture would help...

does the blob flatten out as it approaches the end of the gel?

i've seen gels where the tracking dye runs diffuse until near the end then sharpens to a fine band (it appears to jump to the end of the gel). this appears to be caused by the sample's buffer (not the sample buffer), especially if the sample volume is high. it doesn't seem to effect the results as long as it is permitted to finish migrating.