How do you purify membrane protein? - help (Sep/09/2006 )
I want to express a protein from mycobacterium which is situated on its membrane. I've cloned it in PET 28 and tried to express it with using Lauryl sarcosyl as loading buffer but then also very low amount is coming in supernatent rest all is going in pellet. Is there any way to express these type of protein?
waiting for your help
Perhaps you could try to use Digitonin or Dodecymaltosid, I have used them out of a kit from invitrogen for blue native gels. Have a look on their homepage for the exact procedure. (Sorry, page is inactive at the moment, can't send you the link).
is your protein formed in inclusion bodies, if this is the caswe try to use the ProteoSpin Inclusion body isolation kit from Norgen Biotek. You can obtain your protein from the kit column regardless of your protein fusion. However, having said that you might need to also to use 1% SDS to completlty solublize the membarnes, just to check your protein expression levels (Incubate for 5-10 minutes at room temperature) also, as an altrnative strategy you might need to express your membrnae protein without its membarane signal sequence and thus you can obtain it in a soulble format as you will prevent it from bieng targeted to the membrane!
This is an area that I have extensive experience in and would love to help you in it and other forum members!