Assignment Task
Introduction
Glutathione S-Transferase (GST) is a Phase II metabolic enzyme involved in detoxification of a wide range of chemicals. Identification of GST is performed by western blotting or more easily by enzymatic activity assay.
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The reaction is measured by observing the conjugation of 1-chloro, 2,4- dinitrobenzene (CDNB) with reduced glutathione. This is done by watching an increase in absorbance at 340nm for 5 mins. One unit of enzyme will conjugate 10nmol of CDNB with reduced glutathione per minute at 25°C.
Materials & Samples:
Liver homogenates (pig, lamb and chicken) are provided.
2000μg/ml albumin solution
A 96-well plate
BCA working reagent
Methods
Measurement of the procedure:
Pipette 25μl of each standard or unknown homogenate sample replicate into a 96-well well Add 200μl of the BCA working reagent to each well. Cover plate and incubate at 37°C for 30mins. Measure the absorbance at 570nm on a plate reader.
Measurement of the GST enzymatic activity
1) Make enzyme cocktail:
980μl of potassium phosphate buffer pH 6.5
10μl of 100mM CDNB
10μl of 100mM reduced glutathione
Mix – the solution may be cloudy at first, but should clear up after mixing.
2) Measurement:
For each homogenate sample and a blank, place 900μl of enzyme cocktail into 1.5ml plastic microcuvettes. Incubate at 30°C for 5 mins. To the blank microcuvette, add 100μl Potassium phosphate buffer to enzyme cocktail and zero spectrophotometer. To the sample microcuvette, add 100μl of sample to enzyme cocktail in the microcuvettes and mix. Measure and record absorbance at 340nm for 5 mins (at 0 min and 5 min)
Calculations:
1. Plot the standard curve and get its equation.
2. From its equation, work out the protein concentration for each of homogenate samples and calculate the total amount of protein in 100μl sample (you get concentration for ug/ml, however you used 100ul of homogenate sample for measuring)
3. Calculate the different in absorbance between 0 and 5 minutes and convert it to per minute absorbance
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