Question

Lab: effect of copper (cucl2) on the enzyme activity in bacteria grown on agar plate using LB broth

1) How would you measure the cell viabilty?

2). Prepare a protocol to measure enzyme activity

3). Prepare a brief protocol to excise a protein spot and prepare your sample for analysis by mass spectrometry

Answer 1

1). The cell viability can be determined by the MTT assay:

1). MTT assay -Principle: The MTT assay works by assessing the metabolic activity of the cell. The viability of the cell can be determined by the presence of NAD(P)H dependent cellular oxidoreductase enzymes. These enzymes reduce the MTT dye to an insoluble form, i.e. the purple coloured formazen crystals. The formazen crystals are solubilized in the DMSO to determine their concentration by colorimetrically, which indicates the cell viability. The MTT reduction is high means, the cell is rapidly dividing, low means, slowly dividing.