Asparaginase Assay Kit

Product Name:	Asparaginase Assay Kit (BA0210)
SKU:	BA0210
Detection Method:	Fluorimetric (lambda ex/em = 415/475 nm)
Detection Range:	1.1 - 300 U/L asparaginase
Sample Type:	Serum, plasma, urine and other biological samples
Species Reactivity:	All
Assay Time:	Within 40 minutes
Kit Size:	100 Assays
Equipment Required:	Microplate reader
Storage:	Shipped at room temperature. Store all components at -20 degrees C upon receipt.
Shelf Life:	6 months after receipt
Shipping:	Room Temperature

Asparaginase (EC 3.5.1.1) is a key enzyme in the metabolism of asparagine. Its principal use is as a treatment for acute lymphoblastic leukaemia and lymphoblastic lymphoma where asparagine is an essential amino acid. This kit provides a convenient fluorimetric method to measure asparaginase activity in biological samples. In this assay, o-phthalaldehyde reacts with liberated ammonia where the increase in fluorescence at lambda ex/em = 415/475 nm is directly proportional to enzyme activity.

Safe and fast. Non-radioactive assay. Assay can be completed within 40 minutes.
Sensitive and accurate. Linear detection range 1.1 - 300 U/L asparaginase in a 96-well plate assay.
Convenient and high-throughput. Homogeneous mix-incubate-measure type assay. Can be readily automated to assay thousands of samples per day.

For quantitative determination of asparaginase 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	Prior to the assay, equilibrate all components to room temperature and briefly centrifuge the tubes before opening; the Working Reagent (WR) should be prepared fresh for each assay run.
2	Prepare the enzyme in an enzyme buffer, e.g. 50 mM potassium phosphate, pH 7.4; the protocol is optimised for recombinant E. coli asparaginase and the optimal amount of a different enzyme should be determined experimentally.
3	Dilute serum and plasma samples at least 1:10 and urine 1:50 in water prior to the assay run.
4	Prepare a 1 mM Premix by combining 10 uL of 20 mM Standard and 190 uL of Assay Buffer, then dilute standards per the standard table (1000, 500, 250 and 0 uM).
5	Transfer 10 uL of each standard to a well plus 40 uL of Assay Buffer.
6	Transfer 10 uL of each sample to separate wells of the plate.
7	Prepare enough WR for all sample wells by mixing 2.5 uL of 20 mM Substrate and 45 uL of Assay Buffer for each well.
8	Initiate the reaction by adding 40 uL of WR and incubate the reaction for 20 minutes at room temperature.
9	Add 50 uL of Detection Reagent to all sample wells, tap the plate to mix and incubate for 20 minutes.
10	Measure 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 (per uM) and calculate the asparaginase activity in each sample as: Asparaginase Activity = [(F_Sample - F_Blank) / (Slope (per uM) 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 (20 min) and n is the sample dilution factor. Unit definition: 1 Unit (U) of asparaginase will catalyse the conversion of 1 micromole of the substrate per minute at room temperature and pH 7.4. If sample asparaginase activity exceeds 300 U/L, dilute samples in enzyme buffer and repeat the assay.

Component	Quantity	Storage
Assay Buffer	5 mL	-20 degrees C
20 mM Standard	50 uL	-20 degrees C
20 mM Substrate	300 uL	-20 degrees C
Detection Reagent	5 mL	-20 degrees C