hi,
Iam doing a western for a membrane protein with a mol weight of 36 kda attached with GFP. First I isolate the crude fraction were the membrane is present and then lyse with 20mM beta D maltopyranoside as the detergent to solublize the membrane for two hrs, centrifuge at 14000rpm for 30 min and load the supernatent and the pellet on 10% SDS gel. I do get the protein in the supernatent, but the pellet still shows that the protein is present, which I assume that the detergent has not solublized fully.
Is anyone using/ used this detergent, if so, could you tell me what is the CMC or the optimum concentration for this detergent. Thanks,
jaya
Hi Jaya,
we use dodecyl-beta-D-maltopyranoside to extract entire protein complexes from membranes. We use a concentration that is much lower than 20 mM, however if your protein of interest is a component of a larger protein complex and tightly bound to it, you may spin it down. You can answer this question by using native electrophoresis.
In addition, you could try to use different concentrations of detergent, different types of detergent (CHAPS, triton) or lower the centrifugation speed.
Good luck
Gisela
Hi Jaya,
the cmc of the beta DM is I think 1.7mM but you have to determine the optimum condition that has to be use for your solubilization . The main factor that you have to keep in mind is your detergent to protein ratio. Generally, between 10:1 to 20:1 are good starts. Another thing you can try is also to solubilize overnigth at 4°C if not harmful for your protein.
Good luck
Vanessa
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