I am using the ATP Assay Kit (Colorimetric/Fluorometric) (ab83355) for measuring intracellular ATP from treated cell lines. Although my standards are wokring fine when using the fluorometric approach, my samples are giving negligble redings (almost similar to the background). I am deprotienizing the samples using TCA and then collecting the supernatant, which is used for the assay. Is ATP degradation the issue? or anything else
Dear Muhammad Imran ,
could you provide some details on how you collect the samples and prepare them for the assay?
I'm unfamiliar with the ATP assay you describe - is this based on luciferase activity?
TCA is a fairly strong acid that denatures and precipitates proteins. Since luciferase is a protein, perhaps you need to neutralize the TCA before running the assay.
A third point is that ATP is stabilized by cations, such as Mg+2. Adding Mg2Cl2 may help.
The low pH and deproteinization effect of ice-cold TCA should avoid much of the ATP degradation.
I would look for other sources of problems. For example, do you perform a lysis protocol before deproteinization? In which conditions? Are you sure that you have lysed the cells?
One thing you should check is the expected actual concentration of ATP in these cell lines and take into account the dilutions made until the final reading, maybe it is too diluted.
@carsten Lange
these are cultured epithelial cells (cell line) which are harvested using standard trypsinization. And then suspended innthe ATP assay buffer as provided and recommended with the kit
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