Product Name:	Kinase Assay Kit (Fluorometric) (BA0125)
SKU:	BA0125
Detection Method:	Fluorimetric (λex/em = 530/590 nm), ADP detection
Detection Range:	As low as 0.01 U/L kinase; tolerates up to 300 µM ATP and 10% DMSO
Sample Type:	Purified kinases, cells and other biological samples (user-supplied enzyme, ATP and substrate)
Species Reactivity:	All
Assay Time:	10 minutes detection (plus user-defined kinase reaction, e.g. 30 minutes)
Kit Size:	400 Assays
Equipment Required:	Microplate reader
Storage:	Store all reagents at -20°C.
Shelf Life:	6 months after receipt.
Shipping:	Gel Pack

The kinase reaction produces ADP, which is enzymatically converted to ATP and pyruvate. The pyruvate is quantified by a fluorimetric method at λex/em = 530/590 nm, and the signal is proportional to kinase activity. One reaction volume is 20 µL and the detection is a single working reagent.

Safe, non-radioactive assay.
Sensitive and accurate, quantifying kinase activity as low as 0.01 U/L.
Homogeneous and convenient 'mix-incubate-measure' assay using a single working reagent and 10-minute incubation at room temperature.
Robust and amenable to high-throughput screening, tolerating up to 300 µM ATP and 10% DMSO with Z' factors typically above 0.6, readily automated for tens of thousands of assays per day.

Kinase activity assays and high-throughput screening for kinase inhibitors.
ADP determination in cells and other 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 kit is sufficient for 400 assays in 384-well plates (200 assays in 96-well plates); use black flat-bottom plates, bring all reagents to room temperature and run assays in duplicate.
2	Interference: thiols (β-mercaptoethanol, dithioerythritol, etc.) above 10 µM interfere with this assay and should be avoided.
3	Kinase reaction: users supply their own enzyme, ultra-pure ATP and substrate; set up a 20 µL reaction mixture containing the kinase, ATP and substrate in the provided Assay Buffer (pH 7.0) or a suitable kinase buffer, include a blank control with ATP and substrate but no enzyme, and incubate at the desired temperature for the desired time (e.g. 30 minutes).
4	Standards: prepare 900 µL of 10 µM ADP Premix by mixing 3 µL of the 3 mM Standard with 897 µL distilled water, dilute as described in the standard table and transfer 20 µL of each standard to separate wells.
5	ADP detection: prepare Working Reagent by mixing, per well, 25 µL Reagent A and 25 µL Reagent B, add 40 µL Working Reagent to each assay well, tap to mix and incubate at room temperature for 10 minutes.
6	Read fluorescence intensity at λex = 530 nm and λem = 590 nm.
7	For inhibitor screening, pre-incubate test compounds with the kinase for 10-30 minutes (using a known inhibitor such as staurosporine as a positive control and the compound solvent as a negative control) before adding ATP and substrate to initiate the reaction, then detect the ADP produced as above; the fluorescence decreases in the presence of an inhibitor.

Kinase Activity (U/L) = (ΔF_SAMPLE / (Slope x t)) x (20 µL / Vol), where ΔF_SAMPLE is the fluorescence of the sample well minus that of the blank well, Slope is the slope of the ADP standard curve, t is the kinase reaction time (e.g. 30 min) and Vol is the volume (µL) of kinase added to the 20 µL reaction. If ΔF_SAMPLE exceeds (F_10µM ADP - F_H2O), dilute the enzyme in Assay Buffer, repeat and multiply the result by the dilution factor to ensure the rate is measured at less than 10% ATP conversion.

Component	Quantity	Storage
Reagent A	10 mL	-20°C
Reagent B	10 mL	-20°C
Assay Buffer	25 mL	-20°C
Standard (3 mM ADP)	100 µL	-20°C

Kinase Assay Kit (Fluorometric) (BA0125)

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