Glutathione S-Transferase (GST) Activity Assay Kit (Micro Assay)

Cat. No.: G1506100

GRADE & PURITY BioReagent

Overview

  Glutathione S-transferase (GST) is a crucial Phase II detoxification enzyme in living organisms. It catalyzes the conjugation of reduced glutathione (GSH) to a variety of electrophilic substrates. This kit uses the classic substrate CDNB/BDNB. By measuring the increasing rate of absorbance at 340 nm, the GST activity can be quantified.

  This is a Basic Micro-version Kit that utilizes powder stabilization technology to ensure long-term stability of core reagents. The procedure is optimized for 96-well plates, featuring high-throughput, high sensitivity, and excellent reproducibility. It is particularly suitable for rapid screening and comparative analysis of large sample sizes.

Instruments and Reagents

UV-Vis Spectrophotometer (with 96-well plate reading capability)

Multichannel pipette

Centrifuge, Vortex Mixer

Distilled Water, Anhydrous Ethanol (if needed)

Protein Concentration Assay Kit (e.g., Bradford)

Assay Procedure

Preparation of Working Solutions (Prepare freshly)

1× Assay Buffer: Add 1.5 mL of 10× Assay Buffer to 13.5 mL of distilled water. Mix well to obtain 15 mL of 1× working solution.

4 mM GSH Working Solution: Add 4 mL of 1× Assay Buffer to the GSH powder tube. Vortex to dissolve completely.

2 mM BDNB Working Solution: Add 10 mL of 1× Assay Buffer to the BDNB powder tube. Vortex to dissolve completely. Keep the solution protected from light.

Sample Preparation

Tissues or Cells: Homogenize or lyse samples using ice-cold 1× Assay Buffer. Centrifuge at 10,000g, 4°C for 10 minutes. Keep the supernatant on ice for assay. Determine the protein concentration  of the supernatant using a Bradford assay or equivalent.

Loading and Measurement

Add reagents to a 96-well UV plate according to the table below:

Reagent	Test Well	Blank Well
4 mM GSH Working Solution	25 μL	25 μL
Sample	25 μL	-
1× Assay Buffer	-	25 μL
2 mM BDNB Working Solution	50 μL	50 μL
Total Volume	100 μL	100 μL

Immediately after adding the BDNB working solution, mix by pipetting up and down 3-5 times using a multichannel pipette.

Quickly place the reaction plate into a pre-warmed (37°C) microplate reader.

Immediately initiate kinetic measurement, recording the absorbance at 340 nm every 30 seconds for 5-10 minutes.

Calculation of Results

*Adjusted for Substrate Blank 

     **Using the extinction coefficient 9600 M-1cm-1 

     ***Using the path correction 0.320 cm

     Note: the output of many spectrophotometers is in mOD. Per Well:

• rhGSTP1: 0.005 μg
• GSH: 1 mM
• Substrate: 1 mM

1.  Calculate the Reaction Rate

Use the microplate reader software to calculate the linear slope of absorbance change over time for each well, which is V (ΔA/min).

Corrected V = V (Test Well) - V (Blank Well)

2. Calculate Relative GST Activity

By Protein Concentration: Relative Activity (U/mg prot) = Corrected V (ΔA/min) ÷ Cpr (mg/mL)

By Sample Weight: Relative Activity (U/g) = Corrected V (ΔA/min) ÷ W (g)

One unit of relative enzyme activity (U) is defined as the amount of enzyme that causes a change of 1.0 absorbance unit per minute at 340 nm under the standard assay conditions (37°C) per mg of protein (or per gram of tissue). 

Notes

1. Keep samples on ice during the entire preparation procedure to maintain enzyme activity.

2. It is recommended to use the reconstituted GSH and BDNB working solutions on the same day. Unused solutions can be stored at 2-8°C protected from light for up to 1 week.

3. For accurate results, it is recommended to set 2-3 replicates for each sample.

4. If the initial absorbance of the test well is too high (>1.5) or the reaction curve is non-linear, dilute the sample appropriately with 1× Assay Buffer and remeasure. Multiply the result by the dilution factor.

5. This product is for research use only.

Storage and Shipping

Storage

Store at 2-8°C

Shipped In

Wet ice

Stability And Storage

Each component has a shelf life of 1 year under corresponding storage conditions.