rev.
4/12 For research use only
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:
Gamma Glutamyl Transferase (GGT) Activity Add 90 μl GGT Substrate Mix into each well containing the test samples and positive controls.
Mix well. Do not add to pNA Standards.
Colorimetric Assay Kit
(Catalog # GWB-AXR359; 100 reactions; Store kit at –20oC) 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
I. Introduction:
nm in a microplate reader (A0), incubate for another 30 min to 2 hr at 37°C to measure OD at
The Gamma-Glutamyl Transferase (GGT; EC 2.3.2.2) is an enzyme that transfers gamma- 418 nm again (A1); incubation times will depend on the GGT activity in the samples. We
glutamyl functional groups. It is found in many tissues, the most notable one being the liver,
recommend measuring the OD in a kinetic method (preferably every 3- 5 min) and choose the
and has significance in medicine as a diagnostic marker. GenWay’s Gamma-Glutamyl
Transferase Assay Kit provides a convenient tool for sensitive detection of the GGT in a period of linear range which falls within pNA Standard Curve to calculate the GGT activity of the
variety of samples. The GGT in sample will recognize L-γ-Glutamyl-pNA as a specific substrate samples.
leading to proportional color development. The activity of GGT can be easily quantified
5. Calculation: Plot the pNA standard Curve, then calculate the GGT activity of the test samples:
colorimetrically (λ = 418 nm). This assay detects GGT activity as low as 0.5 mIU.
∆OD = A1 - A0, apply the ∆OD to the pNA standard curve to get B nmol of pNA generated by
II. Kit Contents: GGT in the given time.
GWB-
Components Cap Code Part Number GGT Activity = Sample Dilution Factor = nmol/min/ml = mU/ml
AXR359
GGT Assay Buffer 25 ml WM GWB-AXR359-1 Where: B is the pNA amount from standard Curve (in nmol)
GGT Substrate 1 Bottle NM GWB-AXR359-2 T is the time incubated (in min)
GGT Positive Control 1 vial Green GWB-AXR359-3 V is the sample volume added into the reaction well (in ml)
pNA Standard ( 2mM) 400 µl Yellow GWB-AXR359-4 Unit Definition: One unit GGT will generate 1.0 μmol of pNA per min at 37°C.
Note: One pNA unit ≈ 1.5 IU.
III. Storage and Handling:
Store the kit at -20oC, protected from light. Allow Assay Buffer to warm to room temperature
pNA standard curve GGT positive control and serum sample
before use. Briefly centrifuge vials before opening. Read the entire protocol before performing
the assay. Positive
0.8
Control
IV. Reagent Reconstitution and General Consideration:
0.6 1
GGT Substrate Solution: Add 10 ml assay buffer into substrate bottle and mix well. Take out
O.D 418 nm
O.D. 418 nm
enough substrate solution (90 μl per assay) for the number of assays to be performed in
10serum
experiment. Store the rest of the GGT Substrate Solution into -20°C quickly. Note: The GGT 0.4
Substrate solution is unstable at room temperature (can be hydrolyzed by itself) which 0.5
increases the assay background. 0.2
y = 0.0189x + 0.0302
GGT Positive Control: Reconstitute with 100 µl diH2O. Pipette up and down several times to R² = 0.9974 BK100
0 0
completely dissolve the pellet into solution (Don’t vortex). Aliquot enough GGT Positive
0 10 20 30 40 0 20 40 60
Control (10 µl per assay) for the number of assays to be performed in each experiment and pNA (nmol) Time (min)
aliquot and freeze the rest immediately at -20oC for future use. The GGT Positive Control is
stable for up to 1 month at -20oC after reconstitution or freeze-thaw cycles (< 5 times). Keep
RELATED PRODUCTS:
the GGT Positive Control on ice during the preparation.
ADP/ATP Ratio Assay Kit Ascorbic Acid Quantification Kit
V. GGT Activity Assay Protocol: Glucose Assay Kit Fatty Acid Assay Kit
1. pNA Standard Curve: (Warm for 1-2 min at 37°C to completely melt DMSO): Uric Acid Assay Kit Pyruvate Assay Kit
Add 0, 4, 8, 12, 16, 20 l of the 2 mM pNA standard solution into a 96-well plate in duplicate to Creatine Assay Kit Creatinine Assay Kit
Ammonia Assay Kit Free Glycerol Assay Kit
generate 0, 8, 16, 24, 32, 40 nmol/well standard. Adjust the final volume to 100 l with GGT
Triglyceride Assay Kit Total Antioxidant Capacity (TAC) Assay Kit
Assay Buffer.
Nitric Oxide Assay Kit Glutamate Kit
2. Sample Preparations: GGT Activity Fluorometric Assay Kit Formate Assay Kit
Ethanol Assay Kit Cholesterol Assay Kit
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 FOR RESEARCH USE ONLY! Not to be used on humans.
GenWay Biotech, Inc. Tel: 858-458-0866 | Fax: 858-458-0833
6777 Nancy Ridge Dr, San Diego, CA 92121 USA www.genwaybio.com | sales@genwaybio.com Page 1 of 2
rev. 4/12 For research use only
GENERAL TROUBLESHOOTING GUIDE:
Problems Cause Solution
Assay not working • Use of ice-cold assay buffer • Assay buffer must be at room temperature
• Omission of a step in the protocol • Refer and follow the data sheet precisely
• Plate read at incorrect wavelength • Check the wavelength in the data sheet and the filter settings of the instrument
• Fluorescence: Black plates (clear bottoms) ; Luminescence: White plates ; Colorimeters:
• Use of a different 96-well plate
Clear plates
Samples with erratic readings • Use of an incompatible sample type • Refer data sheet for details about incompatible samples
• Samples prepared in a different buffer • Use the assay buffer provided in the kit or refer data sheet for instructions
• Use Dounce homogenizer (increase the number of strokes); observe for lysis under
• Cell/ tissue samples were not completely homogenized
microscope
• Samples used after multiple free-thaw cycles • Aliquot and freeze samples if needed to use multiple times
• Presence of interfering substance in the sample • Troubleshoot if needed
• Use of old or inappropriately stored samples • Use fresh samples or store at correct temperatures until use
Lower/ Higher readings in Samples
• Improperly thawed components • Thaw all components completely and mix gently before use
and Standards
• Use of expired kit or improperly stored reagents • Always check the expiry date and store the components appropriately
• Allowing the reagents to sit for extended times on ice • Always thaw and prepare fresh reaction mix before use
• Incorrect incubation times or temperatures • Refer datasheet & verify correct incubation times and temperatures
• Incorrect volumes used • Use calibrated pipettes and aliquot correctly
Readings do not follow a linear
• Use of partially thawed components • Thaw and resuspend all components before preparing the reaction mix
pattern for Standard curve
• Pipetting errors in the standard • Avoid pipetting small volumes
• Pipetting errors in the reaction mix • Prepare a master reaction mix whenever possible
• Air bubbles formed in well • Pipette gently against the wall of the tubes
• Standard stock is at an incorrect concentration • Always refer the dilutions in the data sheet
• Calculation errors • Recheck calculations after referring the data sheet
• Substituting reagents from older kits/ lots • Use fresh components from the same kit
Unanticipated results • Measured at incorrect wavelength • Check the equipment and the filter setting
• Samples contain interfering substances • Troubleshoot if it interferes with the kit
• Use of incompatible sample type • Refer data sheet to check if sample is compatible with the kit or optimization is needed
• Sample readings above/below the linear range • Concentrate/ Dilute sample so as to be in the linear range
Note: The most probable list of causes is under each problem section. Causes/ Solutions may overlap with other problems.
GenWay Biotech, Inc. Tel: 858-458-0866 | Fax: 858-458-0833
6777 Nancy Ridge Dr, San Diego, CA 92121 USA www.genwaybio.com | sales@genwaybio.com Page 2 of 2
