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Adjusting pH of cell culture medium for ELISA

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#1 kkmans



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Posted 06 August 2011 - 06:03 AM

Hi everyone,
I would like to measure the testosterone level in TM3 (mouse leydig cell) culture medium using ELISA while this metabolic-active cell can turn the medium yellow in one day. I think the medium should be adjusted back to ~pH 7.4 for the ELISA but I don't know the best way to do so.

I think the simplest way is to neutralize by NaOH, but in some of the assays, I use medium without phenol red, so I have to check the pH of my samples using pH paper one by one. Can I neutralize using buffer in high concentration like Tris-HCl?
For example, add 10 ul 1M Tris-HCl (pH 7.4) to 1 ml medium and assume all samples being adjusted to ~pH 7.4.

Thank you very much!

Edited by kkmans, 06 August 2011 - 06:04 AM.

#2 PAO_ahac



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Posted 16 August 2011 - 02:32 AM

Testosterone is a measured by a competative method where the testosterone-enzyme conj competes with testosterone in the sample for binding sites of the ab which is immobilized on the surface of the microtiter plates. Why not make sure the M of the buffer of the enzyme conjugate is sufficiently strong to overcome the pH of the sample?

You could also premix a known amount of buffer to all samples in the wells prior to adding the conjugate. In any case, you would have to treat the samples and standards in the same manner....

or if diluting the samples, adjust the concentration observed by the appropriate dilution factors used. By this method hopefully, there is no sufficient matrix difference and the curve and samples behave the same...

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