No migration of protein during SDS page.
separating gel - 12% and stacking gel - 5%,
samples taking too long time to come out from the wells and they start migrating from the middle of the well. and after they reach in separating gel no dye front has been seen in the gel. most of the sample remain in the stacking gel. even protein marker also did not show any separation. please suggest what can be the possible factors.
i am using Bio-rad assembly. i changed every buffer and twice and make it fresh every time. but still having same problem.
i am using 4X laemmli buffer having composition:
SDS-8%
mercapto-8%
glycerol-40%
BPB dye - 0.02%
Tris-cl buffer - 250mM
please suggest something.
Mohit: "Something" could be as simple as no current (but voltage shown) when you turned on the power supply, or the wrong pH for stacking gel and running buffer or component(s). Plus, you need 4X loading buffer but the final concentration should be around 1X. Check these basic things and good luck.
thanks Ke-Wei Zhao for ur suggestions. but i checked pH for the buffers. and used fresh running buffer for every time. there may be some problem with power supply. and i used 4x loading dye in vol half of the sample volume.
today i made all reagents and buffers again. and everythng going gud..:)..thanx .
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