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
ConcentrationReagent
10mMTris HCl ph8
150mMNaCl
1mMEDTA
1%NP40
0.1%SDS
1xComplete 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
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