CAn trypsin be used for cell harvest? - (Jan/30/2007 )
Hi,guys,
Currently i'm working on siRNA transfection to Hela cells. After 48 hrs, I want to collect the cells for western blotting.However, the first method i used is using RIPA buffer and scraper to scrape the cells and followed by sonication. This method gives me a pretty low yield which only about 2-3ug/ul. In order for a clear result for my western, i have to increase the concentration of the protein. So i tried using trypsin to trpsinize the cells and spin down them then sonication. Shockingly, this method gives me an even lower yield. Anyone have any idea on that? How can i improve the yield?
By the way, the dish i'm using for transfection is a 3cm one which only have 2.5ml medium. (because the siRNA is expensive, so cant use large dish)
Thx a lot
Den
Currently i'm working on siRNA transfection to Hela cells. After 48 hrs, I want to collect the cells for western blotting.However, the first method i used is using RIPA buffer and scraper to scrape the cells and followed by sonication. This method gives me a pretty low yield which only about 2-3ug/ul. In order for a clear result for my western, i have to increase the concentration of the protein. So i tried using trypsin to trpsinize the cells and spin down them then sonication. Shockingly, this method gives me an even lower yield. Anyone have any idea on that? How can i improve the yield?
By the way, the dish i'm using for transfection is a 3cm one which only have 2.5ml medium. (because the siRNA is expensive, so cant use large dish)
Thx a lot
Den
trypsination with subsequent sonication is the worst you can do as trypsin proteolysis most if not all your cell proteins (this explains your low protein yield); as there are different protocols for RIPA around, I do not know the exact composition of your RIPA; with at least 1% triton x-100 you should efficiently lyse your cells; by the way, your concentration was quite well, but you think the absolute amount of protein is too low; during lysis try to shake vigorously the culture plate on a plate shaker for ca. 20 min
We usually use SDS lysis buffer when we need high protein conc. for western blots. And don't use trypsin.
Good Luck !!!
You can use trypsin if you only use it to detach your cells. However before you go on to lyse your cells you will need to pellet and wash your cells with PBS.
And may be after that to ensure protease resistance add PMSF ( till 1 mM) ? For one hour at room Temp it will suppress serine proteases atack!
I made mammalian cell lysates for western blot by scraping monolayers and pulse sonication + proteases inhibitors. It works good for me!
I think that you should avoid trypsinization if it will be possible