06 December 2016 2 9K Report

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

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