Problem with cell harvest - cell harvest (Dec/03/2007 )
Hi, guys
I am doing some ERK signaling in a time-course treatment of cells in 12 well plate. Since each well of the plate, treating time and factors, is different, I have to harvest them for western blotting one by one. I notice that papers reported scrapping would affect the ERK activation. Could there anyone give me a good suggestion? thanks a lot!
what a guy in our lab does is:
take plate out of incubator.
remove media.
wash with PBS
add RIPA buffer (or what ever you use to make the lysates out of).
collect, spin, boil, load onto gel.
I use SDS buffer, and it works a treat.
could you use trypsin to remove the cells?
V
take plate out of incubator.
remove media.
wash with PBS
add RIPA buffer (or what ever you use to make the lysates out of).
collect, spin, boil, load onto gel.
I use SDS buffer, and it works a treat.
could you use trypsin to remove the cells?
V
Thanks for the reply.
Your way normally works.
My concerns: after collecting sample from one well, just put the plate back to the incubator and wait for the next round?
What i do normally is plan time points such that harvesting time is same for all samples.
Hope this helps.
just like newarray says, just try to have all the samples ready at once.
if you can't, i don't see why you couldn't just put it back in the incubator. of couse, i'd wash the empty well with PBS a few times to remove any trace of SDS or other nasties.
V
as far as trypsin, I think you'll definitely see changes in ERK expression that may not be related to your experiment 
when I did multi-well harvests of adherent cells for phos/non-phos protein panels, it was just as mentioned - stagger the treatment, not the harvest. harvest as quickly as possible with everything as cold as possible
when I did multi-well harvests of adherent cells for phos/non-phos protein panels, it was just as mentioned - stagger the treatment, not the harvest. harvest as quickly as possible with everything as cold as possible
I got it.
Thanks for all!