Passive Lysis Buffer and Westerns

Rob Kirkpatrick kirkpat at cc.umanitoba.ca
Wed Dec 1 10:51:18 EST 1999


Hi all,

I'm working with the Promega Dual-luciferase assay kit so I'm using the
passive lysis buffer to lyse my cells (but doing an active lysis i.e.
scraping).  After scraping, I left the cells in the lysis buffer for 5
min on ice, performed one freeze-thaw (-80, +37) then cleared the
debris.  When I ran out 50 micrograms of protein, I couldn't see my band
by western detection but extracts performed with a different lysis
buffer do give me a band on the same western blot.  My question is:  has
anyone out there used the passive lysis buffer to get protein extracts
for westerns, and if so what was the outcome.  My protein is nuclear so
I'm thinking that I may be pelleting the nucleus and/or my protein.  I
have been able to detect the renilla luciferase that I transfect in, and
I'll be testing the firefly luciferase today and tomorrow.  Any
experience with this will be greatly appreciated.

Thanks,

Rob




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