Eccles:Protein Lysates from Cells in Culture: Difference between revisions
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* Count cells | * Count cells | ||
* Spin cells in 1mL PBS at 4 degrees C, 8000g for 10mins | * 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 do 1x10<sup>5</sup>cells/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. | * Discard supernatant and resuspend cells in cell lysis buffer containing 1x Complete protease inhibitor. I used to do 1x10<sup>5</sup>cells/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... | ||
* Incubate on ice for 30min pipetting up and down occassionally | * Incubate on ice for 30min pipetting up and down occassionally | ||
* Spin debris down at 13000rpm, 5min | * Spin debris down at 13000rpm, 5min | ||
* Aliquot into eppendorfs and store at -80 degrees C (I usually load 7.5uL lysate per well so aliquot slightly more than this per tube) | * Aliquot into eppendorfs and store at -80 degrees C (I usually load 7.5uL lysate per well so aliquot slightly more than this per tube) | ||
Revision as of 17:24, 27 September 2007
Protein Lysates from Cells in Culture
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 MQH2O and filter sterilise.
20x Complete mini Protease Inhibitor
Take 1 tablet and dissolve in 0.5mL MQH2O 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
- 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...
- Incubate on ice for 30min pipetting up and down occassionally
- Spin debris down at 13000rpm, 5min
- Aliquot into eppendorfs and store at -80 degrees C (I usually load 7.5uL lysate per well so aliquot slightly more than this per tube)
