Western blot of apoptotic cells - (Apr/06/2011 )
The procedure I normally use for western blot is as follows: I have the cells in 6-well plates (each well having equal numbers of cells) incubated with the compound of interest for a certain time. Then I wash the cells 2x with PBS buffer before adding lysis buffer and scraping the cells off.
The problem now is that I am testing a compound that is inducing apoptosis in cancer cell lines (MCF-7) in a concentration dependent way. The apoptotic cells (so especially the wells with higher concentration of the compound) will float away and if I wash them I loose them. I only analyse the attached cells not the floating cells. Therefore, I get a weaker signal for the wells with higher concentration of compound.
What is the proper way to retrieve all cells from the 6-well plates? Any advice is much appreciated!
You can collect the lifted off cells in a tube and then lift cells with trypsin (or other agent), spin down, wash in PBS, then spin again etc.
Hmm... after you said it, it seems to be kind of obvious Thank you very much for your help!!