Alcohol Dehydrogenase Enzyme Lab Report

Abstract:

The Yeast alcohol dehydrogenase enzyme (EC 1.1.1.1) belongs to zinc-containing alcohol dehydrogenases family. The aim of this experiment was to determine the subcellular localisation of YAD in S. cerevisiae. The yeast cell was ruptured by homogenisation and fractionated by a process called centrifugation. Protein assay was carried out to calculate the concentration of protein prior to dilutions. ADH assay was carried out to oxidise the ethanol to acetaldehyde and two marker enzymes G6PDH and ALP assays were carried out to aid in the determination of the localisation on YAD. It is concluded that conclude that the ADH enzyme of yeast Saccharomyces cerevisiae that concentrated in the supernatant fraction is located in the cytosol of

Smaller and smaller organelles may be sendimented by successive centrifugation at increasing speed. 1ml of the homogenate (H) was pipetted into a clean microfuge tube labelled P1. The tube labelled ‘P1’ was centrifuged at 1,000g for 10 minutes to sediment the first pellet (P1). The supernatant was carefully removed with an automatic pipette and placed in a micro centrifuge tube labelled ‘P2’. The P2 tube was then placed in the refrigerated centrifuge at a speed of 15,000g for 30-60 minutes at 4°C. 1ml of the homogenisation buffer was added to the P1 pellet and was vortexed to resuspend it. The supernatant was then removed from the P2 tube and placed into a micro tube labelled ‘S’. 1 ml of the homogenisation buffer was added to the P2 pellet and placed on ice. The pellet was then resuspended again by adding small quantity of glass beads and it was vortexed vigorously until the pellet has disappeared from the bottom of the

H and S were diluted 20x and P1 and P2 were diluted 2x to make up a total volume of 1ml each. 50ul of each diluted sample was pipetted into 8 wells of the microplate and 50ul of each protein standard was pipetted into 2 wells. 50ul were incubated with 50ul of alkaline copper reagent. 50ul of alkaline copper reagent was added to every well containing water, buffer, sample or standard and was incubated for 30 minutes. 100ul of Folin reagent was added to the wells and incubated for another 20 minutes. The absorbance was measured at 630-700nm. A standard curve of absorbance vs. protein concentration was plotted and the protein concentration in the diluted sample and the total percentage of activity were