We are checking cancer cell death by activated PBMCs in a co-culture system. We specifically want to analyze the markers (Caspases, PI3K/mTor/AKT, BAX BCL2 and several other markers) using western blot technique. Flow cytometric methods are available but western blot assays are not. Can anyone suggest us the western method to evaluate such pathways of cancer cell killing by activated PBMCs.
Hi Gourav
I suggest for evaluating different markers to do Westernblotting several times. Every time you can stain theòœ membrane with one marker and analyze the staining intensity using Image J free software.
Dear Gourav Chandan ,
I am a little in doubt as to what your main problem is here. I may have some suggestions:
As you have a co-culture assay, you may want to sort the cells and seek differences when they are not grown in this same system, but in a monoculture. So you should label the cell by transfecting the PBMCs and cancer cells with different expressing fluorophore plasmids (e.g., GFP and RFP/mCherry), perform the co-culture, sort them with FACS, and assess the mentioned proteins by immunoblotting.
By this method, you can rule which of these apoptosis-related proteins or PI3K-related pathway may be differently expressed in either population of cells (but in your case, cancer cells, right?). Of course, if you have access to proteomics, you may answer a lot more!
You can stain more than one antibody per membrane if they have very different MW (just cut them prior to staining). Or if you use a membrane for fluorescent staining (it should be specific for that, as it develops bands if much less background), you can multiplex the secondary antibodies with several stainings at a time.
Hope it helps :)
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