Eccles:Protein Lysates from Tissue
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719
Cell Lysis Buffer
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 MQH2 O and filter sterilise.
20x Complete mini Protease Inhibitor
Take 1 tablet and dissolve in 0.5mL MQH2 O by pipetting up and down.
Store on ice.
Dilute 20 fold in cell lysis buffer
Cell Lysis Buffer containing 1x Complete Protease Inhibitor
50uL 20x Complete protease inhibitor
950uL Cell Lysis Buffer
If your tissue is relatively soft and small (eg; mouse kidney, liver, brain etc) then the tissue can be crushed in Cell Lysis Buffer using an eppendorf and pestle on ice.
If you are dealing with larger or fibrotic tissue samples then these are best crushed using a mortar and pestle under liquid nitrogen.
Eppendorf and Pestle Tissue Disruption
- Remove tissue in eppendorf from -80 or liquid nitrogen and store on ice
- Add appropriate volume of ice cold Cell Lysis Buffer containing 1x Complete protease inhibitor (for embryonic mouse kidney 100uL is suitable)
- Crush tissue using pestle until tissue is completely pulverised taking care to keep it cold
- Incubate on ice for 5-10min
- Freeze cells at -80 degrees C
- Thaw cells on ice (these steps help disrupt cell membranes further)
- Spin tissue debris down at 13000rpm, 1min
- Aliquot supernatant protein lysate into usable aliquots and store at -80 degrees C (I usually load 7.5uL of protein lysate per well so aliquot slightly more than this to each tube to completely avoid any freeze/thawing of samples (causes protein degradation))
- Quantitate protein content of lysate (we currently use the BCA kit from Pierce)
Mortar and Pestle Tissue Disruption using Liquid Nitrogen
- Perform crushing in fumehood
- Pour liquid nitrogen into mortar until nearly full (to cool mortar)
- Tip tissue sample into liquid nitrogen
- Start crushing tissue sample with pestle. Can help to tap tissue sample first to break up into smaller fragments. Use circular grinding motion with pestle to crush sample to a powder before the liquid nitrogen completely evaporates.
- Very carefully add a small volume of liquid nitrogen to mortar (very carefully as don''t want crushed tissue to shoot out of mortar)
- Pour crushed tissue and liquid nitrogen into suitable tube and store on ice
- Leave lid off until liquid nitrogen has evaporated
- Add appropriate volume of Cell Lysis Buffer containing 1x Complete protease inhibitor and pipette up and down to thoroughly resuspend tissue
- Incubate on ice for 30mins pipetting up and down occasionally
- Spin tissue debris down at 13000rpm, 1min
- Aliquot supernatant protein lysate into workable aliqots and store at -80 degrees C
- Quantitate protein content of lysate (we currently use BCA kit from Pierce)
