Cell Transformation Group:Protocols/Protein
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Harvesting Cell Lysates for Western Blotting (NEW METHOD)
(Takes about 1-1.5 hours)
- 1. Harvest cells and supernatant into a Falcon tube by trypsinisation
- 2. Use aliquot of supernatant to collect remaining cells in flask or 6-well plate. Transfer to appropriate tube
- 3. Spin for 5-10mins @ 250g (turn on 4°C centrifuge)
- 4. Discard supernatant into Virkon
- 5. Resuspend cells in 1ml of PBS and transfer to a tabbed, eppendorf tube
- 6. Remove a 20µl aliquot for counting
- 7. Spin for 10 mins @ 3000rpm/4°C (get ice)
- 8. Discard supernatant and resuspend in appropriate volume lysis buffer (+1xComplete) to resuspend at 2 x 104 live cells/µl or other useful concentration depending on expt
- 9. (can do this now or later) Lyse cells for 30mins on ice, mix, spin cell debris down, transfer supernatant to labelled eppendorf
- 10. Store at –20°C
Cell Lysis Buffer | |
---|---|
Concentration | Reagent |
10mM | Tris HCl pH8 |
150mM | NaCl |
1mM | EDTA |
1% | NP40 |
0.1% | SDS |
1x | Complete protease inhibitor |
Add Complete just prior to use
Nb – Complete is stored at 25x concentration
Add:
- 40μl 25x to: 1ml volume
- 2μl 25x to: 50μl volume