G6PDH Inhibitor Screening Kit (BA0194)

Product Name:	G6PDH Inhibitor Screening Kit (BA0194)
SKU:	BA0194
Detection Method:	Non-radioactive colorimetric assay based on the reduction of the tetrazolium salt MTT in an NADPH-coupled enzymatic reaction to a reduced form of MTT with an absorption maximum at 565 nm. The increase in absorbance at 565 nm is proportional to enzyme activity, and percent inhibition is determined by comparing treated versus untreated G6PDH.
Sample Type:	Purified G6PDH reactions; test compounds dissolved in the solvent of choice (e.g. DMSO or DMF)
Species Reactivity:	All
Assay Time:	30 minutes
Kit Size:	100 Assays
Equipment Required:	Microplate reader
Storage:	Store all components at -20°C upon receipt.
Shelf Life:	6 months after receipt
Shipping:	Room Temperature

This assay measures G6PDH inhibition through the reduction of the tetrazolium salt MTT in an NADPH-coupled enzymatic reaction. As G6PDH reduces NADP to NADPH while oxidising glucose-6-phosphate, the coupled reaction converts MTT to a reduced form absorbing maximally at 565 nm. The increase in absorbance at 565 nm is proportional to enzyme activity, and percent inhibition is calculated by comparing the activity of G6PDH treated with a test compound against untreated enzyme.

Safe. Non-radioactive assay.
High-throughput. Homogeneous mix-incubate-measure type assay that can be readily automated on an HTS liquid-handling system.
Rapid and reliable. Can be completed in 30 minutes with no 37°C heater required.

High-throughput screening for inhibitors and evaluation of glucose-6-phosphate dehydrogenase inhibitors.

Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Step	Procedure
1	The assay is based on a kinetic reaction; addition of the Working Reagent should be quick and mixing brief but thorough. Assays can be run at any desired temperature (e.g. 25°C or 37°C).
2	Sample preparation: for L. mesenteroides G6PDH, dilute purified G6PDH to 0.0003 U/µL using dH2O. For other species, experimentally determine the Km and adjust the substrate volume so the final substrate concentration in the 100 µL reaction is near the Km. Dissolve test compounds in the solvent of choice; if using DMSO/DMF, first test enzyme tolerance (for L. mesenteroides G6PDH, keep the 5 µL compound addition at <=2 v/v% DMSO or <=40 v/v% DMF).
3	Transfer 20 µL of G6PDH into separate wells.
4	Reserve two wells with G6PDH for the Blank (no substrate) and Control (no inhibitor).
5	To the Control and Blank wells, add 5 µL of the solvent in which the test compounds are dissolved (e.g. 5 µL 2 v/v% DMSO).
6	To the remaining G6PDH wells, add 5 µL of the test compounds. Tap plate and mix.
7	Incubate the plate for 15 minutes at 25°C.
8	Prepare 1x Substrate by diluting 10x Substrate 10-fold in dH2O (8 µL 1x Substrate per reaction well).
9	Prepare Reaction Mix per well (except the Blank) by mixing 70 µL Assay Buffer, 8 µL NADP/MTT, 1 µL Diaphorase and 8 µL 1x Substrate. Prepare Blank Reaction Mix per Blank well with 70 µL Assay Buffer, 8 µL NADP/MTT, 1 µL Diaphorase and 8 µL dH2O (no substrate). Add 75 µL Blank Reaction Mix to the Blank well and 75 µL Reaction Mix to the remaining wells. Tap to mix briefly and thoroughly.
10	Incubate the plate for 15 minutes at room temperature and read optical density at 565 nm.

% Inhibition = (1 - (dOD_TestCpd / dOD_NoInhibitor)) x 100%, where dOD_TestCpd is the OD_565nm of a test compound minus the OD_565nm of the Blank well at 15 min, and dOD_NoInhibitor is the OD_565nm of the Control minus the OD_565nm of the Blank well at 15 min.

Component	Quantity	Storage
Assay Buffer	10 mL	-20°C
Diaphorase	120 µL	-20°C
NADP/MTT	1 mL	-20°C
10x Substrate (450 mM G6P)	100 µL	-20°C