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Help- lysing cells with triton x-100


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#1 mejoiningtoo

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Posted 02 May 2011 - 11:40 PM

hi guys,

I want to lyse fibroblast to measure intracellular ATP. I saw a paper using 1% triton x-100 as lysing buffer, incubated at 37C for 30 mins in 48-well plate, and measure ATP in lysate. Could I just take the liquid from the plate using pipette after the 30min? Or do I need to scrape cells off from the plate, centrifuge and take the supernatant?

Thanks a lot!

#2 KevinK

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Posted 04 May 2011 - 06:21 AM

In our experience at Promega, intracellular ATP is extremely labile in cell lysates. With our original systems, we recommend a TCA extraction to eliminate ATPases and stabilize the sample. We have ready-to-use ATP assays (CellTiter-Glo) that are much more convenient. The chemistry in this assay is designed to stop ATPases as well. In summary, Triton x-100 is NOT enough to keep the ATP from degrading and I highly recommend looking at alternative protocols.

Kind Regards,
Kevin
Promega Corporation
Madison, WI




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