Harvest of cells from 96 well plates for Western blots - (Jun/22/2006 )

Hi- I would like to do some 96 well assays (RNAi) followed by verification that my target is inhibited in expression- does anyone have a good method for getting the cells off? Trypsinization? Thanks in advance-

Cover with RIPA buffer, put on a slow shaking platform (what you use for WB) for 15 min and recover with a pipette. You can also do it directly in Laemmli

Can u get enough sample to test expression with western blot?

I was trying to do the same but in 24 well plate and always had problems with getting enough samples. I can see downregulation in my samples compared to the control but even the positive control is only weakly detected and with the RNAi treated one its even lower. So its difficult to come to any conclusion. I am therefore trying out the same experiments in 6 well plates.

Right, I can't see how it is possible to get enough protein from 55X less cells than I would use for a 60mm dish and then make accurate protein determinations to load equivalent protein in each lane of the gel. Maybe I'll try replicates of 10, then run 5X less protein than I usually do on the Western (50ug down to 10ug)

scolix can you probe your blot for a housekeeping gene like actin and show there is equal amount of that in each condition? (if there is, there may not be)

Biorad (and others as well I guess) sell kits for protein determination in low amount proteins samples. You can use that to determine your protein concentration. You can load the whole amount of the 96 well (it's risky) and probe with an anti-actin or other HK gene product and compare the lanes using a program as many people do on this forum. I loaded 5ug total proteins on gels and I got very nice results. It depends on the antibody and on the abundance of your protein, but it's not impossible. You can give it a try and lat me know. Some of my friends were working with drugs and they were treating the cells in 96 wells and using the extract for WB

dnafactory,

does it have to be RIPA buffer? RIPA contains Triton X (I think) but the cell lysis buffer I use has NP40 in it. Will either detergent removr tyhe cells from the wells?

Thanks