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Using protein content to standardize ELISA data?


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2 replies to this topic

#1 zienpiggie

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Posted 15 August 2011 - 09:56 PM

Hi there,

I have been doing a bunch of ELISA to detect enzyme activities, as well as glutathione content in cell lines. For each of these data, it seems to be very common to express the enzyme activity relative to the protein content. Same goes for the glutathione content. What I found however is that my treatment seems to increase protein content of cells.

I am wondering therefore, is it possible to have increased protein content without affecting cell number? If that is the case, what is the rationale of using protein content to standardize certain data such as the total glutathione content?

Any input will be appreciated..

#2 prodes

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Posted 16 August 2011 - 09:34 AM

Hi zienpiggie,

Do you test overexpressed protein for different durations or are you working with different proteins? In case you overexpress different proteins even if you normalize them by cell number you will most likely get different total protein mass affected mainly by the expression level of the protein. Therefore, while not perfect normalization, this would reflect the enzymatic activity you test (I presume coming from the overexpressed protein) over its concentration variation from sample to sample.

Cheers

#3 zienpiggie

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Posted 24 August 2011 - 01:25 PM

Hi prodes, no I am not over expressing any proteins, at least not intentionally. After looking back at my data, it seems to me that my treatment does have an effect on cell proliferation, in that it does increase cell number. That is likely why I am seeing an increase in cell protein. I am running an experiment to confirm this. Nevertheless, it therefore make sense to normalize to protein content.

Thank you for your input.




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