Lysis of CHO and HEK Mammalian Cells for SDS-PAGE - (Aug/06/2001 )
I need a protocol for lysis of my CHO and HEK adherent cells for use in SDS-PAGE and immunoblotting. Is there a general protocol for this, or will I need specific lysis buffers for my different cell lines?Thanks, Allison.
I think that the best way is to trypsinize them, spin them and then resuspend them in SDS-PAGE loading buffer.
Why not rinse them with PBS, scrape them into PBS with a cell scraper, pellet them, and bring up in SDS-sample buffer.
A problem is the DNA released from nuclei which may be sheared with a tuberculin syringe ... but this is a hassle.
The "DNA snot" problem can be avoided by:a) breaking cells first with 1% NP-40 with 2 mM spermidine present followed by spinning out nucleior b) breaking cells first with 1% NP-40 followed by enzymatically destroying DNA with bensonase (or DNAase I).
I wash the Hek in PBS and then add 1x sample buffer directly on the cells. Scrape to collect the solution and sonicate with a tip sonicator a few seconds to break the DNA. Is easy to pipet then. SM
I used trypsin before, but need to be careful if your protein is has extracellular region which contains Arginine as trypsin can cleave it off. This is ok normally but if your primary antibody is targeting the cleaved region, you won't get any signal.