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How to dissociate multimers

multimers dissociation heteromers dissociate

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#1 labmonkey

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Posted 07 September 2011 - 08:09 AM

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

#2 mdfenko

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Posted 09 September 2011 - 07:26 AM

triton will displace sds from the protein. you should reduce it before treating with sample buffer.
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Also tagged with one or more of these keywords: multimers, dissociation, heteromers, dissociate

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