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Amplite® Colorimetric Gama-Glutamyltransferase (GGT) Activity Assay Kit
Example protocol
AT A GLANCE
Prepare the test samples, GGT positive control, and the serially diluted pNA standards (50 μL).
Add the GGT working solution (50 μL).
Incubate for 10-30 minutes at 37 °C.
Measure the absorbance at 418 nm.
Thaw all the kit components at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTIONS
Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles
Reconstitute the GGT Positive Control (Component F) with 100 µL of ddH2O to make a 500 µg/mL GGT Positive Control stock solution. Mix well by pipetting and store at -20 °C.
Note: Must be used within 1 month of reconstitution. Avoid freeze/thaw cycles.
PREPARATION OF STANDARD SOLUTIONS
https://www.aatbio.com/tools/serial-dilution/11801
PREPARATION OF WORKING SOLUTION
Reconstitute the GGT Substrate (Component B) by adding 5 mL of the GGT Assay Buffer (Component A). Mix thoroughly until well combined. Protect the mixture from light and store it at -20°C.
Note: Avoid freeze/thaw cycles.
Tissue and cells can be homogenized using the GGT assay buffer. To remove any insoluble material, centrifuge the homogenized sample at 13,000xg for 10 minutes. Serum samples can be added directly to the wells without any prior treatment. Adjust the volume of all samples to 50 µL by adding GGT assay buffer.
SAMPLE EXPERIMENTAL PROTOCOL
Prepare one or more GGT positive control samples along with the test sample. The recommended concentration is 20 µg/mL in GGT Assay Buffer. For example, for a 20 µg/mL positive control, add 20 µL of the GGT positive control stock solution to 480 µL of GGT Assay Buffer.
Table 1. Layout of pNA standards and test samples in a 96-well solid black microplate. (STD = pNA Standards (STD1-STD7, 3.125-200 µM), BL= Blank Control, TS = Test Samples.)
BL | BL | Positive Control | TS |
STD 1 | STD 1 | ... | ... |
STD 2 | STD 2 | ... | ... |
STD 3 | STD 3 | ||
STD 4 | STD 4 | ||
STD 5 | STD 5 | ||
STD 6 | STD 6 | ||
STD 7 | STD 7 |
Table 2. Reagent composition for each well.
Well | Volume | Reagent |
STD 1-STD 7 | 50 µL | Serial Dilutions (3.125 to 200 µM) |
BL | 50 µL | GGT Assay Buffer |
GGT Positive Control | 50 µL | GGT Positive Control |
TS | 50 µL | Test Sample |
Prepare pNA standards (STD1-7), blank controls (BL), GGT Positive Control, and test samples (TS) as outlined in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
Add 50 µL of GGT Working Solution to each well containing the pNA standard, blank control, GGT Positive Control, and test samples. For a 384-well plate, add 25 µL of GGT Working Solution to each well instead.
Incubate at room temperature for 10-30 minutes, protected from light.
Monitor the absorbance intensity with an absorbance microplate reader at 418 nm.
References
Authors: , and Lambré, Claude and Barat Baviera, José Manuel and Bolognesi, Claudia and Cocconcelli, Pier Sandro and Crebelli, Riccardo and Gott, David Michael and Grob, Konrad and Lampi, Evgenia and Mengelers, Marcel and Mortensen, Alicja and Rivière, Gilles and Steffensen, Inger-Lise and Tlustos, Christina and Van Loveren, Henk and Vernis, Laurence and Zorn, Holger and Herman, Lieve and Roos, Yrjö and Aguilera, Jaime and Andryszkiewicz, Magdalena and Cavanna, Daniele and Kovalkovicova, Natalia and Liu, Yi and di Piazza, Giulio and Chesson, Andrew
Journal: EFSA journal. European Food Safety Authority (2024): e8509
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Journal: Endocrine (2024): 511-518
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Journal: Romanian journal of internal medicine = Revue roumaine de medecine interne (2024): 203-209
Authors: Shao, Zhenyong and Xu, Yuyan and Zhang, Xuebang and Zou, Changlin and Xie, Raoying
Journal: Strahlentherapie und Onkologie : Organ der Deutschen Rontgengesellschaft ... [et al] (2024): 523-534
Authors: Harrer, Philip and Inderhees, Julica and Zhao, Chen and Schormair, Barbara and Tilch, Erik and Gieger, Christian and Peters, Annette and Jöhren, Olaf and Fleming, Thomas and Nawroth, Peter P and Berger, Klaus and Hermesdorf, Marco and Winkelmann, Juliane and Schwaninger, Markus and Oexle, Konrad
Journal: EBioMedicine (2024): 105007
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