I have been making a cerebellar lysate from adult mouse using the following buffer:
3mM NaHEPES (pH 7.5), 320mM Sucrose, 0.1% Triton, PMSF, protease inhibitor tablets. We are finding the low salt concentration important for maintaining protein solubility.
I have some surprising results which I think can possibly be explained by a lack of ER proteins in our lysate. Our protein has no EndoH sensitivity and is not present in a non-glycosylated form, most unexpected. Similarly, we are getting very low yields of a protein we expect to be completely retained in the ER.
Does anybody have any advice about these buffer conditions and how to best solubilize both ER and plasma membrane proteins in low salt, or of course any alternative explanations.
Thanks very much
Coomb RaiderMember Since 11 Mar 2010
Offline Last Active Jan 09 2013 04:22 AM
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