When I run the protein samples with the marker on SDS-PAGE gel everything went well till the end of the gel, after transferring the gel on the nitrocellulose membrane and staining with poceaous I found that the protein stuck on the half the gel while the marker completed till the end.
The first time I thought that the problem is the Agarose gel that I used to seal the bottom and top slide as it was over than 2%. So I removed it and It went well and got a good result , then I changed the gel composition because one of my components was run out and I begin to use 1.5 Molar tris HCL 8.8 instead of 3Molar and again I got the same result of the first one the protein stuck at the half. 4th time I tried to get 3M tris HCL and return to the old composition of the gel and the same.
I couldn't determine where the problem exactly.
Check carefully if you had sufficient SDS and B-mecapthoethanol in your sample buffer. Alternatively, run an SDS PAGE with the same marker and protein sample after checking your sample buffer, stain the gel and destain and compare the profile. From your blot it seems your protein are degraded prior to SDS PAGE because i can't see distinct bands on the blot.
I hope my suggestion may help gain you time.
It might be due to temperature of the gel. Usually it happens in winters when the room temperature is around 15 C. Before preparing gel, bring all the reagents at 37 C by keeping them in the water bath ( except APS and TEMED). It also happens when your APS goes bad. I suggest to work with 1.5 TrisHcl 8.8 pH ( check pH of both the buffers before making a gel, adjust them but keep in mind that too much of salt could hamper the protein run). Try to keep your assembly ( poured gel into glass plates) near warm place ( between 25 c - 35 C, not more then that). Make a fresh aliquot of APS, enough for one week ( store at 4 C).
All the best.
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