I want to prove a kind of protein in medium is from exocytosis but not lysed dead cells. I can detect the protein in medium by ELISA(no band in Western blotting), but I'm not sure where does the protein come from. By what method, I can detect if there are lysed cells in medium? I've tried to detect β-Actin and GAPDH with WB and get no band. Is that OK? Is there any other reliable assays?
Many use a colorimetric assay for lactate dehydrogenase (LDH), which is a soluble and stable enzyme found inside every living cell. You'll have to search the literature for a suitable assay.
Sure, absence of GAPDH is a first hind that there are no dead cells .However this is ever a question of sensitivity of a qulaitative method (WB). I would agree that colorimetric assay-based analyses mentioned above may be an alternative way. Morover, it is possible to determine via fluorenscense-based assays (e.g by an AV/PI assay + FACS analysis) whether or not there is anpoptosis/cell death .
Thomas J Raub Thanks a lot. I'll try to find some literature and try some assays about LDH.
Andreas Eisenreich Thanks a lot. I'm also worrying about the sensitivity of WB. I think we will try GAPDH ELISA along with LDH.
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