Dear Urmila,

Please read the following text:

Gamma Glutamyl Transferase (GGT) Activity

Colorimetric Assay Kit

GGT Activity Assay Protocol:

1. pNA Standard Curve: (Warm for 1-2 min at 37°C to completely melt DMSO):

Add 0, 4, 8, 12, 16, 20 l of the 2 mM pNA standard solution into a 96-well plate in duplicate to generate 0, 8, 16, 24, 32, 40 nmol/well standard. Adjust the final volume to 100 l with GGT

Assay Buffer.

2. Sample Preparations:

Tissues (10 mg) or cells (1×106) can be homogenized in the 200 l GGT Assay Buffer then centrifuged (13,000 x g, 10 min.) to remove insoluble material. Serum samples (10 l) can be directly added into each well. Prepare test samples to 10 μl/well with GGT Assay Buffer in a 96-well plate. We suggest testing several doses of your sample to make sure the readings are within the linear range of the standard curve.

3. Reaction Mix:

Add 90 μl GGT Substrate Mix into each well containing the test samples and positive controls.

Mix well. Do not add to pNA Standards.

4. Measurement: For pNA Standard Curve, measure OD at 418 nm in a microplate reader. For the samples and positive controls, incubate the mix for 3 min at 37°C, then measure OD at 418 nm in a microplate reader (A0), incubate for another 30 min to 2 hr at 37°C to measure OD at 418 nm again (A1); incubation times will depend on the GGT activity in the samples. We recommend measuring the OD in a kinetic method (preferably every 3- 5 min) and choose the period of linear range which falls within pNA Standard Curve to calculate the GGT activity of the samples.

5. Calculation: Plot the pNA standard Curve, then calculate the GGT activity of the test samples:

∆OD = A1 - A0, apply the ∆OD to the pNA standard curve to get B nmol of pNA generated by GGT in the given time.

GGT Activity = B/TV x Sample Dilution Factor = nmol/min/ml = mU/ml

Where: B is the pNA amount from standard Curve (in nmol)

T is the time incubated (in min)

V is the sample volume added into the reaction well (in ml)

Unit Definition: One unit GGT will generate 1.0 μmol of pNA per min at 37°C.

Note: One pNA unit ≈ 1.5 IU.

For more details and to view figures, please see attached file.

Hoping this will be helpful,

Rafik

thank you sir for reply......but tell me that  is it also calculated with molar extinction coeffecint?

For calculating the molar extinction coefficient of the indicated wavelength, please see attached file.

Rafik