Does anyone have any experience with NEM (N-Ethylmaleimide) in a nuclear extraction?
I am trying to stabilize the SUMO modifications of a protein during my nuclear extraction for a western blot.
Should all three of my buffers (cell membrane lysis, nuclear lysis, sample buffer) contain NEM?
Are there any special conditions that are required for the crosslinking?
Hi,
When I was working on a DUB during my thesis we did not add NEM to the sample buffer but I'd keep an inhibitory concentration of NEM throughout the process in the 2 extraction buffers.
I don't think you can have a quick deSUMOylation between the moment the NEM of your final nuclear extract is diluted in the Laemmli sample buffer and the denaturating action of bME/DTT and SDS it contains.
I'd say that if you efficiently inhibit deSUMOylating enzymes you don't need to crosslink anything, the SUMO moieties will remain covalently bound to the lysines side chains of the SUMOylated proteins
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