I'm using 100 mM phosphate buffer (7.5), NADH final concentration of 0.13 mM. and sodium pyruvate in phosphate buffer to a final concentration of 2.3 mM.
the ratio as the following:
933.3 ul NADH
33.3 ul Pyrovate
33.3 ul sample (cell culture medium)
I need equation to calculate the enzyme activity for example in mU/ml.
PS.
Can I use normal flate microtiter plate or this need specific plate since this in UV range?
Hi,
Firstly, the LDH assay measures the oxidation of NADH as you are following the reduction in absorbance as NADH is converted to NAD+ at 320 nm which will correlate to LDH activity. When we do this assay in the lab, we usually will follow the decrease in absorbance over time and not take a "snapshot" measurement. If you are using this assay to measure cellular toxicity, you will need to calculate the ratio of LDH activity in the cell vs. the cell culture medium. This is done by measuring the rate of decrease over time to generate the slope. Once you have the slope multiply by your dilution factor for your samples.
I hope this helps.
http://www.clinchem.org/content/19/7/766.full.pdf+html
http://link.springer.com/article/10.1007%2FBF02388199#page-1
yes you will need plates which will work in UV range
Hi,
Firstly, the LDH assay measures the oxidation of NADH as you are following the reduction in absorbance as NADH is converted to NAD+ at 320 nm which will correlate to LDH activity. When we do this assay in the lab, we usually will follow the decrease in absorbance over time and not take a "snapshot" measurement. If you are using this assay to measure cellular toxicity, you will need to calculate the ratio of LDH activity in the cell vs. the cell culture medium. This is done by measuring the rate of decrease over time to generate the slope. Once you have the slope multiply by your dilution factor for your samples.
I hope this helps.
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