Lysing hela cells - poor protein recovery (May/29/2006 )

I'm lysing some non-transfected hela cells. All I want from them is the protein, non-denatured. I'm lysing with Tris, NaCl and NP-40. I am getting what I think is a low amount of protein from them. I lysed five confluent 75cm^2 flasks and only got ~2mg of protein. That doesn't seem like much to me. Other cells I have used (High Five) give me 2mg from a single 25cm^2 flask.

Could it be the lysis buffer is ineffecient at lysing hela's or is this a realistic amount of protein to get?

this doesn;t seem like much to me - could you please give us more info? are you pipetting your lysis solution directly onto the cells or resuspending a pellet? if straight onto cells, you might want to try a rubber policeman as well to lift the cells a bit....

I'm trypsinizing the cells, spinning down, adding the lysis buffer and then vortexing.

what do you use as protease prevention?

PMSF, aprotinin and leupeptin.

doesn't the trypsin mess you up here?

I thought you had to scrape the cells to uplift for protein prep or you would get poor results?

but I have only worked with primary keratinocytes and confess that I don't know for sure

hi
HeLa cells can support trypsin without big effects. SO i don't suppose the problem came from the cell detachement process per se. But if trypsinization is done too long (and maybe too strong, try to dilute it to 1:5) thzcan decrease protein recovery.

Hmm.

I might try scraping then, see if I get better results.

one assumes you are keeping everything - EVERYTHING - on ice during the extraction? that will also increase yield pretty dramatically...avoid freeze/thaws of your extracts...etc...do you know this stuff already?