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My question concerning this assay, is as follows. Since the amount of protein loaded per well in addition to the standards is diluted ½, then the actual amount of 3NT per amount of protein loaded is actually 3nt from standard curve/2xstarting protein.
For example, if I loaded 50ug protein in the well and the result was 10ng/ml 3nt, the actual concentration in my loaded sample would really be 10ng/ml 3NT in 25ug protein loaded because of the initial dilution with 2x buffer.
Asked on Apr 10 2015
As explained in the protocol page 17 (https://www.abcam.com/ps/products/113/ab113848/documents/ab113848%20Nitrotyrosine%20ELISA%20Kit_24%20June%202014_JC%20(website).pdf) the final concentration and molarity of the standard dilution series takes into account the 2x dilution, so you do not need to do an additional 2x dilution adjustment with your sample OD readings. Thus, in your example the original sample concentration of 3NT in the sample would be 10 ng/ml.
Kevin HansonAbcam Scientific Support
Answered on Apr 10 2015