So I'm learning that in order to increase the yield of membrane proteins by biotinylation and strep-magnetic bead pull-down, I must make sure nuclei is intact after lysis, therefore using mechanical lysis method is better than solution method.
I'm just wondering why I need to make sure nuclei is intact.
Adam B Shapiro
One possibility that occurs to me is that if you break open the nuclei and release all the nucleic acids, you could end with with a viscous extract that will be difficult to work with.
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Gary S. Laco
just use a mild detergent lysis method (0.5% NP40, Tris 20 mM pH 7.4, 100 mM NaCl), gently mix every 2-3 min. at 5oC for 10-15 min. then centrifuge at low speed to pellet the nuclei. The membrane proteins will be in the supernate.
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