cell lysis buffer to use directly on the dish - (Oct/28/2008 )
Hi everybody!
I need some advice; i'm looking for cell lysis buffer to be used directly on culture dishes. Collegue recomended me a procedure using Laemmli lysis buffer (glycerol, tris, sds, ph6.8). Basically, u add the buffer on culture dish, scrape the cells (in my case hepatocytes) and freeze in liquid nitrogen. Upon thawing u sonicate the lysate (50 pulses) and proceed directly with Bradford assay for determination of protein concentration. What concerns me is that no protease inhibitors r added and the lysates r not centrifugated to remove cellular debris. And what do u think about it? any ideas or maybe alternative procedures? I will just add that im optimizing some technique and im using 35mm dishes with around 550.000 cells so former procedure is very convinient; i wont lose proteins due to not efficient scraping or subsequent centrifugation but can i really trust it???
Thanks a lot for your help!!!
I need some advice; i'm looking for cell lysis buffer to be used directly on culture dishes. Collegue recomended me a procedure using Laemmli lysis buffer (glycerol, tris, sds, ph6.8). Basically, u add the buffer on culture dish, scrape the cells (in my case hepatocytes) and freeze in liquid nitrogen. Upon thawing u sonicate the lysate (50 pulses) and proceed directly with Bradford assay for determination of protein concentration. What concerns me is that no protease inhibitors r added and the lysates r not centrifugated to remove cellular debris. And what do u think about it? any ideas or maybe alternative procedures? I will just add that im optimizing some technique and im using 35mm dishes with around 550.000 cells so former procedure is very convinient; i wont lose proteins due to not efficient scraping or subsequent centrifugation but can i really trust it???
Thanks a lot for your help!!!
There are plenty of buffer for cell lysate. Try the protocol part of this website, also the papers regarding the type of protein you are working with. The important part is: what is your purpose? What are you using the lysate for? If you don't want to denature your protein, don't use Laemmli buffer. Also, the high concentration of detergent is likely to interfere with your concentration reading.
About your questions: I assumed that the Laemmli lysis buffer you are talking about is the loading (or sample buffer) for SDS gel without dye. If so, there is no need for protease inhibitors, the SDS would denature every proteins anyway. the lysate will be quite viscous. The sonication may help a little but you will have to spin after that anyway.