Eccles:Protein Lysates from Cells in Culture: Difference between revisions
(New page: =Protein Lysates from Cells in Culture= ===Cell Lysis Buffer=== 5mL 0.1M Tris HCl pH 8 (10mM)<br> 0.44g NaCl (150mM)<br> 0.02g EDTA (1mM)<br> 0.5mL nonidet P40 (1% w/v)<br> 0.05g SDS (0.1...) |
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0.05g SDS (0.1% w/v)<br> | 0.05g SDS (0.1% w/v)<br> | ||
Make up to 50mL with MQH<sub>2</sub>O and filter sterilise.<br> | Make up to 50mL with MQH<sub>2</sub>O and filter sterilise.<br> | ||
Store at 4 degrees C.<br> | |||
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). | |||
===20x Complete mini Protease Inhibitor=== | ===20x Complete mini Protease Inhibitor=== | ||
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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... | ||
Confluent 6 well (9.6cm<sup>2</sup>) lyse in 150uL cell lysis buffer.<br> | |||
Confluent T75 lyse in 800-1000ul cell lysis buffer.<br> | |||
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) | |||
Latest revision as of 15:55, 12 October 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.
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).
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...
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)
