Question

BioChemistry Lab question:

Your PI would like to measure the concentration of alanine aminotransferase (ALT), a liver enzyme, in a homogenate of mouse liver. Your lab already has a bottle of ALT and a very specific and selective antibody that is known to bind to ALT and ALT only. What technique will you use to measure ALT in the liver homogenate? Why choose this/these technique/s? Briefly describe the results you would obtain from each technique.   

This is a lab question and so far in the class, we have done immunoblot, electroblotting, ELISA, Bradford Coomassie assay, protein ladder, SDS-PAGE, and Column chromatography.

Answer 1

ALT and AST, two liver enzymes are frequently use to check the liver health, in human we use serum for determination of these enzymes. As specified in the statement specific reagent for ALT and specific antibody for ALT is also present so, qualitatively and quantitative we can estimate the levels of this enzyme in liver homogenate. You can use ELISA reader to see the enzymatic activity through time kinetic (activity of this enzyme in one min) and an immunoblot would be suitable for the same (western blotting). For measurement of enzymatic activities you need to determine protein concentration in the homogenate is also must. Similarly for western blotting, you need to run homogenate sample over SDS-PAGE for separations of protein subsequently transfer of protein on to membrane.

Please note that higher activity of this enzyme through ELISA or Western blotting will suggest liver inury or hepatotoxicity.