Low Protein Content In Cell Lysate - (Feb/07/2013 )

Hi

I am using MDA-231 cell for preparing my lysate, and i have been using RIPA from Santacrz Biotech however over time and again i have been getting very less protein content in range of 5-10 micro grams/micro liter

i usually do lysis of around 4 X 10⁶ cells in around 300 mico liter of RIPA also while lysisng then i vortex them after 10 mins for 1 hr and also pass them through a syringe 28 gauge yet i find some pellet at the bottom after i spin down at 14000 rpm for 15 min ( which i ideally belive i should not be getting)

your comments are welcome

Thanks

-do you add protease inhibitor?
-after addition of RIPA and resuspension by vortexing, rotate more than 1 hr at 4C

btw, 5-10ug/ul is not really that low.