These cells were differentiated in 24-well plate and I need to perform a western blot looking for a membranous protein (PARK7). I'm using the following procedure but it's not working.
1- 5 days post-differentation, wash the cells with PBS then trypsinize
2- move cells to eppendorfs (each eppendorf cointains cells of 1 well), add 50 microliters of RIPA lysis buffer with protease inhibitor cocktail
3- place on ice for 15 min
4- centrifuge at full speed for 30 min at 4C, discard lipid layer on top and debris on the bottom
I gave details just to make sure no step is missing.
Thank you in advance,
Norma.
Normah, what protein are you looking for? I'd skip the trypsin step (which might destroy proteins on the extracellular side of the plasma membrane) and lyse the cells directly in the dish.
Also check the pellet (will happily dissolve in Laemmli buffer, but will need some shearing to reduce DNA viscosity)
I'm looking for the Parkinson Protein 7 (PARK7). So I'll lyse the cells in the dishes this time.
Thank you Wolfgang Schechinger!
Is recombinant PARK7 protein commercially available? Could it be used as an internal spike during the RIPA lysis to be sure the entire procedure is working?
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