Alpha-Mannosidase Activity Assay Kit (BA0014)

Product Name:	Alpha-Mannosidase Activity Assay Kit (BA0014)
SKU:	BA0014
Detection Method:	Colorimetric kinetic (OD 405 nm)
Detection Range:	1 to 250 U/L (10 µL sample, 10 min reaction)
Sample Type:	['Plasma', 'Serum', 'Tissue', 'Culture media']
Species Reactivity:	All
Assay Time:	10 minutes
Kit Size:	100 Assays
Equipment Required:	Microplate reader
Storage:	4°C
Shelf Life:	6 months after receipt
Shipping:	Room Temperature

Alpha-mannosidase (AMA) is an enzyme which catalyses the cleavage of the alpha form of mannose. Alpha-mannosidase assists in the breakdown of complex sugars from glycoproteins in the lysosome. Defective or deficient AMA activity causes alpha-mannosidosis and leads to deterioration of the central nervous system in children. This non-radioactive, colorimetric AMA assay is based on the cleavage of 4-nitrophenol from a synthetic substrate. Nitrophenol becomes intensely coloured after addition of the stop reagent, and the increase in absorbance at 405 nm after addition of the stop reagent is directly proportional to the enzyme activity.

Fast and sensitive. Linear detection range (10 µL sample): 1 to 250 U/L for a 10 minute reaction.
Convenient and high-throughput. Homogeneous mix-incubate-measure type assay that can be readily automated on HTS liquid handling systems for processing thousands of samples per day.

Alpha-mannosidase activity determination in biological samples (e.g. plasma, serum, tissue and culture media).

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	Prepare samples: serum and plasma can be assayed directly. For tissue, rinse in phosphate buffered saline (pH 7.4) to remove blood, homogenise 50 mg in approximately 200 µL buffer containing 50 mM potassium phosphate (pH 7.5) and centrifuge at 10,000 x g for 15 min at 4°C, using the supernatant. For cell lysate, collect cells by centrifugation at 2,000 x g for 5 min at 4°C, homogenise or sonicate in cold 50 mM potassium phosphate buffer (pH 7.5) and centrifuge at 10,000 x g for 15 min at 4°C.
2	Equilibrate Substrate Buffer to the desired reaction temperature (e.g. 25°C or 37°C). Prepare standards by mixing 10 µL of 12.5 mM Nitrophenol standard with 490 µL dH2O to make a 250 µM standard, then dilute as described in the standard curve table.
3	Transfer 100 µL of each standard into wells of a clear flat-bottom 96-well plate.
4	Transfer 10 µL of each sample into separate wells, add 90 µL Substrate to each sample well and tap the plate briefly to mix.
5	Incubate at 25°C (or the desired temperature) for 10 minutes, add 100 µL of Stop Reagent to each well and tap the plate briefly to mix.
6	Read OD405nm.

Subtract the blank OD (water, #4) from the standard OD values and plot the change in OD against standard concentrations to determine the Slope. AMA Activity = [(OD_Sample - OD_Blank) / Slope] x n (U/L), where OD_Sample is the OD_405nm value for each sample, OD_Blank is the OD_405nm value of water (standard #4) or the sample blank, Slope is from the linear regression of the standard points and n is the dilution factor. Unit definition: 1 Unit (U) of AMA will catalyse the conversion of 1 µmole of 4-nitrophenyl-alpha-D-mannopyranoside to 4-nitrophenol and alpha-D-mannose per min at 25°C and pH 4.5.

Component	Quantity	Storage
Substrate Buffer	10 mL	4°C
Stop Reagent	12 mL	4°C
Standard (12.5 mM Nitrophenol)	1 mL	4°C

Alpha-Mannosidase Activity Assay Kit (BA0014)

Description