Hi,
I'm trying to purify couple of integral membrane proteins from Sf21 cells. Typically I isolate total membrane (PM, ER, golgi etc) after cell lysis and couple of centrifugations. Additionally I also wash the membranes with 0.5-1M NaCl several times to remove membrane associated soluble proteins. Now I want to isolate PM fractions only because of two reasons. 1) I am purifying few contaminating proteins that don't come off after Ni and anion exchange step. 2) I want to enrich PM only to reduce the cost of detergent I am using for initial solubilization. There are several sucrose gradient protocols but if someone already used those for insect cells or have modified working protocol please share.
Thanks