| Steven Finkbeiner, Departments of Neurology and Physiology, UCSF
http://gweb1.ucsf.edu/labs/finkbeiner/Protocol/protocol_list/Sub_Fract.htm
Prepare buffer A:
10 ml buffer A
50 ul PMSF (1/200)
50 ul Benzamidine (1/200)
20 ul Leupeptine (1/500)
50 ul Aprotinin (1/200)
Wash and scrape cells. Procedures may vary in this step based on the cell type and what is needed for the final samples.
Spin cells at 4 C for 2 min at 2000 rpm
Wash quickly in buffer A solution
Spin again at 4 C for 2 min at 2000 rpm
Resuspend cells in 200 ul buffer A solution
Leave on ice for ~20 min
Aspirate up and down with a 1 ml syringe/25 gauge needle 15 times
Check sample on microscope with trypan blue to verify that fractionation has
taken place.
Spin at 4 C for 3 min at 3000 rpm
Pipette out supernatant (cytoplasm) and transfer to new eppendorf tube
Resuspend pellet (nuclei) in buffer A solution and spin again at 4 C for 3
min at 3000 rpm repeating this step for a total of 3 times, discarding the
supernatant each time
Resuspend pellet in 50 ul buffer A
Sonicate all samples and take the OD as stated in "Western Blot Protein
Preparation" protocol
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