Which protocol do you use?

I once prepped membranes from COS7 and other cells using this protocol:

Buffer A

250 mM sucrose

1 mM EGTA

10 mM HEPES-KOH (0.11915)

Protease inhibitors

pH 7.5 using KOH

Buffer B

1M Na2CO3

Buffer C

250 mM sucrose

1 mM MgCl2 (0.010165)

10 mM HEPES-KOH

-wash cells, scrape, resuspend in buf. A (approx 0.5 mL per 6w plate well)

- homogenize (Dounce-homog. 25x or -20°)

- centrif. 2x 500 x G 15 min 4°C (keep supernatant)

- supernat. + 100 µL buf. B per mL buf. A

- shake 45 min 4°C

- 100.000 x G 15 min 4°C

- resuspend pellet in buf C

never had a problem with this protocol...

In fact, membrane would not dissolve in water. That's why you can collect it by centrifuge. You might want to sonicate it to disperse micell or dissolve membrane by detergents such as triton, chaps, NP-40, etc.

If proteins in/on your membrane are causing this, you might want to add some salt (NaCl, KCl, etc) because salt dissociate interaction by charges. Detergents are also effective for this.

And you might also want to check if the pellets not dissolved are membrane you want. It may be cytoskelton or some kind of debris.

Thanks a lot.The buffer I have been using has 125mM NaCl, 1.33mM MgSO4, 4.83mM KCl , KH2PO4, CaCl2, HEPES, Abscorbic acid and glucose

your buffer has too much salt Prerna. Too much salt denatures proteins and makes them less soluble. Try HEPES alone. If you need to add protease inhibitors, first make sure they will not interfere with other analysis you are trying to do such as quantifying protein or Western blots. Hope it helps.

I would try the Stephan protocol minus the buffer B. And yes, if it is membranes, it won't be soluble unless you add detergent.