Non-Neuronal Adherent Cells-Elvis

1.            Place plates on ice.  Remove media (can save for LDH, etc.).

2.            Wash 1X with cold PBS. Place 1-1.5mL PBS on cells and scrape. Transfer to 1.5mL eppendorf tube.

3.            Centrifuge at 4°C, 200rpm, 10min

4.            Remove PBS and add 50-100µL lysis buffer (bigger pellet = bigger volume)

5.            Maintain constant agitation for 30min at 4°C.

6.            Centrifuge at 12,000 rpm and 4°C for 20min.

7.            Place tubes on ice and transfer 80µL to 0.6mL eppendorf for WB and save remainder in original eppendorf for protein.

8.            Save in -80°C

Non-Neuronal Loosely Adherent Cells  

1.            Place 100mm dishes on ice.  Scrape with existing media and transfer to conical.

2.            Pellet at 200rpm, 4°C, 10min.

3.            Resuspend pellet in 1mL cold PBS and repellet at 200rpm, 4°C, 10min.

4.            Add 50µL lysis buffer to each tube and transfer to 1.5mL eppendorf tube.  Ultrasonicate each sample to 10sec. Place on ice on shaker at 300rpm for 15min. 

5.            Centrifuge at 12,000 rpm and 4°C for 20min.

6.            Remove as much supernatant as you can in 0.6mL eppendorf for WB.  Leave ~10µL in original eppendorf for protein. 

7.            Save in -80°C

Hi Lesley,

This is a method that we've used (adapted from the method described by Karsdal et al. (2003) J Biol Chem 278: 44975–87) on differentiated RAW 246.7 cells (volumes for 24 well plates).

* RIPA buffer recipe that I use:

50 mM Tris-HCl pH 7.4, 150 mM sodium chloride, 1% Nonidet P40, 0.25% sodium-deoxycholate, 1 mM ethylene diamine tetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, 1 μg/ml each of aprotinin, leupeptin and pepstatin, 1 mM Phenylmethylsulfonyl fluoride, PMSF and 5 mM of Ethylenediaminetetraacetic, EDTA, added to chelate calcium.

Hope this helps,

Wei

Thanks everyone. I think I may have cracked it now!