How to dissociate multimers - (Sep/07/2011 )
Hi,
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?
RIPA buffer:
150mM NaCl,
1% tritonX,
0.5% NaDeoxycholate,
0.1% SDS,
50mM Tris, pH 8.0
plus Roche complete without EDTA
Sample buffer (1x conc):
62.5mM Tris pH 6.8,
10% glycerol,
2% SDS,
5% BME,
0.002% Bromo blue
triton will displace sds from the protein. you should reduce it before treating with sample buffer.