Neuraminidase Activity Assay Kit (Colorimetric or Fluorometric) (BA0135)

Product Name:	Neuraminidase Activity Assay Kit (Colorimetric or Fluorometric) (BA0135)
SKU:	BA0135
Detection Method:	Colorimetric or Fluorometric
Detection Range:	0.1 to 10 U/L (colorimetric); 0.01 to 2 U/L (fluorometric) at 37 degrees C
Sample Type:	Biological samples
Species Reactivity:	All
Assay Time:	60 minutes
Kit Size:	100 Assays
Equipment Required:	Microplate reader
Storage:	-20 degrees C
Shelf Life:	6 months after receipt
Shipping:	Gel Pack

This kit measures the N-acetylneuraminic acid (NANA) released by neuraminidase in one step. The change in colour intensity of the reaction product at 570 nm or fluorescence intensity at 585/530 nm is directly proportional to neuraminidase activity in the sample.

Sensitive and accurate with a linear detection range at 37 degrees C in 96-well plate of 0.1 to 10 U/L for colorimetric assays and 0.01 to 2 U/L for fluorometric assays
Simple and convenient homogeneous assay requiring only two absorbance measurements and completed in 60 minutes
High-throughput assay that can be readily automated as a 96-well plate assay to screen thousands of samples per day

Direct measurement of neuraminidase activity in biological samples
Drug discovery evaluation of neuraminidase 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	Equilibrate all components to the desired reaction temperature (i.e. 37 degrees C) and prepare a 400 uM Standard Premix by mixing 20 uL of the 10 mM Standard and 480 uL distilled water, then dilute the standard as described in the dilution table.
2	Transfer 20 uL standards into separate wells of a clear flat-bottom 96-well plate, and transfer 20 uL of each sample into two separate wells, one for sample activity and one for the sample blank.
3	Immediately prior to starting the reaction, prepare enough Working Reagent for all sample and standard wells by mixing per reaction tube 30 uL Assay Buffer, 55 uL Substrate, 1 uL Cofactors, 1 uL Enzyme and 0.5 uL Dye Reagent; for the sample blank wells substitute 55 uL Assay Buffer for the 55 uL Substrate.
4	Add 80 uL of the appropriate Working Reagent to each well.
5	Incubate the reaction plate protected from light at 37 degrees C for 20 minutes and measure the OD at 570 nm, then incubate for a further 30 minutes protected from light and measure the OD again; for the fluorometric procedure use a black 96-well plate and measure fluorescence at 530/570 nm.

Plot the OD or F measured at 50 minutes for each standard against the standard concentrations and determine the slope by linear regression. Subtract the 20 minute values from the 50 minute values for the sample, sample blank and H2O reactions, then Neuraminidase Activity = (delta RSAMPLE - delta RBLANK - delta RH2O) / Slope x (1/t) (U/L), where Slope is the slope of the standard curve in uM-1 and t is the reaction time between readings (30 minutes). One Unit (U) of neuraminidase will catalyse the release of 1 umole of NANA per minute under assay conditions.

Component	Quantity	Storage
Assay Buffer	6 mL	-20 degrees C
Substrate	6 mL	-20 degrees C
Cofactors	120 uL	-20 degrees C
Dye Reagent	60 uL	-20 degrees C
Enzyme	120 uL	-20 degrees C
Standard	500 uL	-20 degrees C