5mL 0.1M Tris HCl pH 8 (10mM)
0.44g NaCl (150mM)
0.02g EDTA (1mM)
0.5mL nonidet P40 (1% w/v)
0.05g SDS (0.1% w/v)
Make up to 50mL with MQH2O and filter sterilise.
Store at 4 degrees C.
This cell lysis buffer has a limited shelf life (at 4 degrees C). I recommend making fresh buffer up annually. My own personal experience has been that after ~18months my protein lysates began appearing very distorted following SDS PAGE and were no longer usable (whether fresh lysates were made from >18month old buffer or lysates that had been stored at -80 for >18months were used).
Take 1 tablet and dissolve in 0.5mL MQH2O by pipetting up and down.
Store on ice.
Dilute 20 fold in cell lysis buffer
50uL 20x Complete protease inhibitor
950uL Cell Lysis Buffer
Harvest cells into 15mL tube by typsinisation as usual
Spin at 250g for 5min
Aspirate off supernatant
Resuspend cells in 1ml PBS
Spin at 250g for 5min
Aspirate off supernatant
Resuspend cells in 1mL PBS and move to an eppendorf
Take 20uL of cells for counting
Count cells
Spin cells in 1mL PBS at 4 degrees C, 8000g for 10mins
Discard supernatant and resuspend cells in cell lysis buffer containing 1x Complete protease inhibitor. I used to do 1x105cells/uL but can do whatever you want. By making lysates at a set concentration of cells/uL you don''t need to quantitate the protein in the lysates. Simply load equal volumes of lysate to get equal loadings of protein. Popular choices by 2nd floor researchers of the department were...
Confluent 6 well (9.6cm2) lyse in 150uL cell lysis buffer.
Confluent T75 lyse in 800-1000ul cell lysis buffer.
If you choose this way you will need to quantitate your protein lysates to enable loading equal total cellular protein amounts onto your gel. I have followed this method and have found it to work well.
Incubate on ice for 30min pipetting up and down occasionally
Spin debris down at 13000rpm, 5min
Aliquot into eppendorfs and store at -80 degrees C (10ul aliquots usually helpful)
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