I am having difficulties dissociating ion channel heteromers in my Western Blots. I lyse my cells (293Ts) one ice with RIPA buffer. I find that the cells lyse within 2-5 mins and I simply use a pipette to wash the dish and transfer the cells to an eppy. I then incubate the lysate on a roller for 1h and spin down 20 mins at 15,000 x g.
I dilute the samples in RIPA buffer and add a basic laemelli sample buffer before boiling the samples at 70oC for 10 min (also tried 95oC for 5min) and load 20ug on a tris/glycine gel.
I find that I have very specific binding (absolutely no x-reaction with neg controls, even other members of the channel family), but that I have a weak monomer band and then a blur of multimer bands leading to a super ugly giant black smear.
Does anyone have any recommendations for dissociating the multimers or has anyone encountered this issue before?
50mM Tris, pH 8.0
plus Roche complete without EDTA
Sample buffer (1x conc):
62.5mM Tris pH 6.8,
0.002% Bromo blue
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