We are now performing the ACC test in our lab following the Penrose method and I have some questions about the assay. I use this protocol

Protocol to prepare a-ketobutyrate St curve

A stock solution of 100 mM a-ketobutyrate is prepared in 0.1 M Tris-HCl pH 8.5 and stored at 4 °C. Just prior to use, the stock solution is diluted with the same buffer to make a 10-mM solution from which a standard concentration curve is generated. Each in a series of known alpha-ketobutyrate concentrations is prepared in a volume of 200 microl and transferred to a glass test tube (100x13 mm); each point in the series is assayed in duplicate. Three hundred microl of the 2,4-dinitrophenylhydrazine reagent (0.2 % 2,4-dinitrophenyl-hydrazine in 2 N HCl; Sigma- Aldrich Co.) is added to each glass tube and the contents are vortexed and incubated at 30 °C for 30 min during which time the a-ketobutyrate is derivatized as a phenylhydrazone. The color of the phenylhydrazone is developed by the addition of 2.0 ml of 2 N NaOH; after mixing, the absorbance of the mixture is measured at 540 nm.

How can you convert the α-ketobutyrate mM (1-0.05 mM) to micromol? Tha graph reporteted micromoles

How much is your ABS values for each concentration (1-0.05 mM)?

Thank you in advance

Patrizia