Question

Biochemistry Question

You are studying the enzyme Holidase (53 kDa) and want to measure its kinetics (its reaction rates). Holidase catalyzes the conversion of the substrate SPBRK into the small molecule product VCTN. Neither SPBRK or VCTN are colored, which means you cannot measure those concentrations directly, but you do have a polyclonal Gt anti VCTN antibody as well as a monoclonal Ms anti VCTN antibody which you hope will be useful. What antibody technique that we covered in class would be most helpful for quantifying the amount of VCTN produced? a. Western Blot b. ELISA C. Co-Immunoprecipitation

Answer 1

Out of these ELISA technique we will use to measure kinetics of enzyme reaction . This is a quantitative method which is widely used in medical field.

For the estimation we can use sandwich ELISA. In which we will take antibody coated plate ( against VCTN) . After that we will add the reaction mixture. VCTN present in reaction binds with antibody and rest of the reaction mixture will be washed out. After this step anti VCTN antibody tagged with HRP will be added and last HRP substrate will be added. There is production of colour which will be directly propotional to amount of VCTN in the reaction.