Glutaminase Assay Kit

Product Name:	Glutaminase Assay Kit (BA0218)
SKU:	BA0218
Detection Method:	Fluorimetric, kinetic; read at lambda ex/em = 415/475 nm
Detection Range:	0.066-50 U/L glutaminase
Sample Type:	Serum, plasma, urine, biological samples
Species Reactivity:	All
Assay Time:	Within 50 minutes
Kit Size:	100 Assays
Equipment Required:	Microplate reader
Storage:	Store all components at -20 degrees C upon receipt.
Shelf Life:	6 months after receipt
Shipping:	Room Temperature

Glutaminase (EC 3.5.1.2) is a key enzyme in the metabolism of glutamine. Glutamine is an important amino acid in the metabolism of cancer cells, and increased glutaminolysis has been linked to cancer. This kit provides a convenient fluorimetric method to measure glutaminase activity in biological samples. In the assay, o-phthalaldehyde reacts with liberated ammonia, and the increase in fluorescence at lambda ex/em = 415/475 nm is directly proportional to enzyme activity.

Fast and safe: the assay can be completed within 50 minutes and is non-radioactive.
Sensitive and accurate: linear detection range is 0.066-50 U/L glutaminase in a 96-well plate assay.
Convenient and high-throughput: homogeneous mix-incubate-measure type assay that can be readily automated to assay thousands of samples per day.

Quantitative determination of glutaminase activity in biological samples.

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	This assay is based on a kinetic reaction, so addition of the Working Reagent to samples should be quick and mixing brief but thorough; use of a multi-channel pipettor is recommended and the assay can be run at room temperature.
2	Prior to the assay, equilibrate all components to room temperature and briefly centrifuge tubes before opening; the Working Reagent should be prepared fresh for each assay run.
3	Prepare enzyme in an enzyme buffer such as 20 mM potassium phosphate, pH 7.4; the protocol is optimised for glutaminase from Bacillus amyloliquefaciens, and for other enzymes experimentally determine the optimal amount per well.
4	Dilute serum and plasma samples at least 1:10, and dilute urine 1:50 in water prior to the assay run.
5	Prepare a 1 mM Standard Premix by combining 10 microlitres of 20 mM Standard with 190 microlitres of distilled water, then prepare all standards according to the standard preparation table and transfer 10 microlitres of each Standard to separate wells of a black 96-well plate.
6	Transfer 10 microlitres of each sample to separate wells of the plate.
7	Prepare enough Working Reagent for all assay wells by mixing 5 microlitres of 20 mM Substrate and 45 microlitres of Assay Buffer for each well.
8	Initiate the reaction by adding 40 microlitres of Working Reagent to all assay wells, tap the plate to mix and incubate for 30 minutes at room temperature.
9	Add 50 microlitres of Detection Reagent to all wells, tap the plate to mix and read for 20 minutes, measuring fluorescence intensity at lambda ex/em = 415/475 nm.

Subtract the blank value (Standard #4) from the standard values and plot delta-F against the standard concentrations. Determine the slope (uM-1) and calculate glutaminase activity as: Glutaminase Activity = [(F_Sample - F_Blank) / (Slope (uM-1) x t)] x n (U/L), where F_Sample and F_Blank are the measured fluorescence values of the sample and blank, t is the reaction time (30 min) and n is the sample dilution factor. Unit definition: 1 Unit (U) of glutaminase will catalyse the conversion of 1 micromole of L-glutamine per minute at room temperature and pH 7.4. Note: if sample glutaminase activity exceeds 50 U/L, dilute samples in enzyme buffer and repeat the assay.

Component	Quantity	Storage
Assay Buffer	5 mL	-20 degrees C
20 mM Substrate	500 uL	-20 degrees C
20 mM Standard	50 uL	-20 degrees C
Detection Reagent	5 mL	-20 degrees C

Glutaminase Assay Kit (BA0218)

Description