proein sample - help (May/13/2009 )

hello, everyone.
i am focusing on the apoptotic effect of virus on tumor cell recently,but there is a problem during my preparation of protein sample:
the virus strongly induce cell apoptosis,a lot of cell detach from the dish and it seems many are lysised(i can see debris looks like sands under mircroscope).so do i need collect all the cells,including dead cell,or just collect those alive ?
first,if i collect all the cell with a higher speed,3000rpmX3min for example,is it likely that the cells were split during centrifuge, what may lead to the loss of protein in successive wash with cold PBS. because cell treated with virus for different time show apoptosis vary in degree, the protein concentration in different sample may different.these make the result less convinence.
second,if i collect alive cell only(some people advise me to do),the adherence cell only, i collect cell different in NO. (the longer treated with virus,the less cell adherent to dish or keep its integrity in structure.) according to different sample.these also lead to the difference in totall protein among different sample.
which procedure shall i choose? or is there a better one ? i wish someone give me some suggestions, i am a beginner.
by the way, i need to detect cleaved caspase-3,(A549 Cell),does the cell express caspase3 high level? do i need prepare protein sample with high concentration. please make a example.

Collect the live cells, the dead ones have lost the virus and also are lysed and so the proteins are degrading. Base your western loading on cell number (10,000 - 100,000 cells/ul should be enough) rather than total protein.

Cleaved caspase 3 will only be present when the cells are apoptosing, all cells that apoptose should have it. A549 certainly do.

bob1 on May 13 2009, 06:06 PM said: