I was measuring ATP concentration in E. coli for one of my research project. I saw some literature mentioning ATP concentration in E. coli as 1-5mM. However, I am getting much less (in micromolars). I was wondering what is the experience of others. I tried cell lysis by both acid lysis method and then neutralizing as well as I tried sonicating. I am using luciferase assay kit to moniter the luminescence for the assay.
I have not done the luciferase assay by using E.coli, but I have a lot of experience of doing the assay with human myoblasts (whole cellular lysate) which are ATP-rich cells. My results were nanomoles per microgram of protein. My friend did the assay using fibroblasts and he ended up nanomoles as well. It sounds a bit unlikely to get 1-5mM.. Of course the value depends on normalization. I normalized my values using µg of protein. I used the assay provided by Molecular probes (Life technology) and measured the luminescence with Fluostar Omega platereader and Bradford assay for protein concentration. This may not be helpful but I think your scale is more reliable than mmoles.
How are you calculating the intracellular ATP concentration from the ATP concentration in the extract? Have you confirmed thorough cell disruption by microscopy?
What is your measuring process? Can you please share that along with your reference measurements?
Thank you all of you for your response. I was using a kit from Biovision for the assay. I did not expect that phosphate buffer will interfere the results. When I was trying to figure out everything that might affect, I noticed that addition of even 5mM phosphate buffer in the reaction mixture decreases the signal by half. For the concerns about cell lysis I have tried the sonication which dissolves almost everything also which did not affect the result. For now I am thinking it was the phosphate that was inhibiting the signal.
Dear Dr. Shapiro I was converting it to intracellular concentration by considering the cellular volume I took and adjusting all the dilution factors I did.
You also have to consider than only part of the volume of a cell pellet consists of the cytoplasmic volume of the cells. Much of the volume consists of extracellular fluid, cell membranes, and periplasm.
https://bionumbers.hms.harvard.edu/bionumber.aspx?&id=100011&ver=9
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