Hello there,
We are trying to assess hepatic damage via the quantification of alanine aminotransferase (ALT) activity in murine tissue samples. In order to do so, we are using Sigma's ALT activity assay kit to determine the activity (https://www.sigmaaldrich.com/catalog/product/sigma/mak052?lang=es®ion=ES&gclid=CjwKCAjw1f_pBRAEEiwApp0JKPd4Lj_22BOK8dfQUTgdrO_vMfo5IIcWSAJSnqTTmh4x259XtGv-bRoCDRcQAvD_BwE ).
This kit allows the determination by a -non specified- coupled reaction with pyruvate (ALT product), that results in a colorimetric/fluorometric product (not indicated). The rate at which this chromophore is produced, is proportional to the activity of the latter transaminase.
We have developed the assay following the colorimetric method, but we have run into a problem. Our pyruvate standards do not rest stable during the whole assay, therefore, we cannot plot a specific calibration curve that correlates absorption with pyruvate concentration.
We have followed the same procedure that Sigma establishes, but as you can see in the image provided, the results do not make sense. The signal for each pyruvate standard increases with time, when it should remain relatively identical.
¿Any clue as to why this event is happening?
Pd: Our plate reader doesn't allow measurements at 570 nm, so we are measuring at 560 nm.
Hello, did you check the pH of reaction mixture. May be it differ from optimum pH range for your enzyme.
Hello Karen A. Darbinyan ,
No we have not thought about it. As it's a comercial kit we believe that all those variables come predefined. We will check! Thanks for the answer.
Any other ideas?
Hi, were you able to solve this issue? We are usign the same kit and I'm not quite sure we understand how to calculate the sample values
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