Scraping or Trypsinization for westerns?? - (Aug/11/2005 )
Hi All,
I am detecting some phospho proteins in MCF-7 cell lysates. My question is : Is it better to scrape the cells or trypsinize them for Westerns?? I normally pull of the media and scrape them in cold 1X PBS. Someone told me that trypsinization is better..
Whatcha opinion??
I am detecting some phospho proteins in MCF-7 cell lysates. My question is : Is it better to scrape the cells or trypsinize them for Westerns?? I normally pull of the media and scrape them in cold 1X PBS. Someone told me that trypsinization is better..
Whatcha opinion??
i think it depends on what are you looking at. i usually scrape cells just like you in cold PBS, and its ok. however for some experiments you want your cells to remain intact, when with scrapping there is a big chance youll mash some of them. trypsinization is a solution, but might be that it initiates some signaling issues, which you dont want either. i know that for the adherent cells you can also try just to dump few ml of PBS with EDTA, wait a bit, and then just wash them off the plate and spin.
Hey Pria,
I had this same exact question because I wanted to ensure my phospho protein was not dephosphorylated in the time it took me to get my attached cells off the dish. I had been using trypsin and it took about 8 min. I decided to compare cell scraping in PBS at 4 C with using trypsin at 4 C. Scraping was obviosely faster. I compared samples taken under the same conditions, same extraction buffer, etc, but different attachment procedure. I probed for both the phospho protein (erk1/2pp) and later stripped the membrane and probed for actin. I developed the two blots on the same film and found that the signal for both actin and phospho protein extracted from samples detached via trypsin was much stronger than that detected from the scraped samples (about 2 orders of magnitude!).
I did not attempt to use a BCA test or similar to quantify total amount of protein because I was really interested in the state of the protein rather than the total amount. I found the ratio of phosphorylated protein to actin didn't really change between cell detachment methods.
I only tried scraping this one time, so maybe others have tricks to get higher yeilds.
If you go with trypsin as I have, make sure you neutralize it before lysing the cells.
thanks xorb for the reply...
one more question...
after you added trypsin...did you let it sit or did u swirl it around the plate and pull it off....Also if I let it sit in theplate....will adding PBS do the neutralization bit??? thanks again...Also my timepoints are 5 mins after tretment....will the trypsinization time (in ur case 8 mins) affect it??
one more question...
after you added trypsin...did you let it sit or did u swirl it around the plate and pull it off....Also if I let it sit in theplate....will adding PBS do the neutralization bit??? thanks again...Also my timepoints are 5 mins after tretment....will the trypsinization time (in ur case 8 mins) affect it??
I add trypsin at 4 C and keep the samples that way for about 6-8min. After that, I "wash" the cells off the plates by aspirating the trypsin in and out of a pipett. PBS will not neutralize trypsin. You must use either a serum containing media or trypsin neutralizing solution. This is actually quite important as trypsin will eat up your protein once the cells are lysed and it is in solution. Even the protease inhibitor cocktails that are recommended for protein sample storage will not be enough to counter the trypsin you are adding.
As far as the time affecting your samples, I recommend you do a control. Keeping cells at 4 C is supposed to theoretically prevent phosphatases from deactivating your protein. If you have some doubt, I recommend doing the control sooner than later. You should be able to answer that question with one simple experiment and than put the issue out of your mind.
Hope this helps
hi
i get same results by tripsinisation or scrapping regarding total protein extracts.
for extraction in which you need to separate nuclear prot from cytopasmic prot, i woulnd surely not recommend scrapping.
btw for trypsinisation : i put it 5' at 37, add complete medium (inactivation of trypsin) and gently detach cells by pipetting. After pelleting i wash with cold PBS.
5 or 8 minutes of trypsinisation willnot that affect your extraction, but if you analyzes a protein tough with cells environment, i would recommend to trpysinize not more than 5', and do the same time of treatment each time.
Enjoy cell biology 
fred
we can't let us being bored by cells! 
thanks fred...
forget about cells boring me....its driving me nuts :-)
hi guys,
everyone else in my lab (where i've just moved) scrapes cells with a pipette tip in RIPA lysis buffer directly in the plate.... i however wash with cold pbs, add trypsin to the cells for 5 mins at 37C and then inactivate with a similar amount of complete media. i then extract using a triton-x lysis buffer with phosphatase inhibitors and protease inhibitors in for 1 hour at 4C... my stuff seems to work ok but try getting my colleagues to change their mind!!!!!
everyone else in my lab (where i've just moved) scrapes cells with a pipette tip in RIPA lysis buffer directly in the plate.... i however wash with cold pbs, add trypsin to the cells for 5 mins at 37C and then inactivate with a similar amount of complete media. i then extract using a triton-x lysis buffer with phosphatase inhibitors and protease inhibitors in for 1 hour at 4C... my stuff seems to work ok but try getting my colleagues to change their mind!!!!!
Scraping is the standard method. It's faster, saves your time, money on trypsin, and is more efficient. It's not like your growing the cells on is it
Rhombus
Agree.
And as long as your using phosphatase inhibitors, you'll be okay.