Cat.No.:	Kit-0869
Product Overview:	The assay is initiated with the enzymatic hydrolysis of the glucoside by α-Galactosidase. The enzyme catalysed reaction products p-nitrophenol, can be measured at a colorimetric readout at 400 nm.
Description:	Alpha-galactosidase is a glycoside hydrolase enzyme that hydrolyses the terminal alpha-galactosyl moieties from glycolipids and glycoproteins. It is encoded by the GLA gene. Two recombinant forms of alpha-galactosidase are called agalsidase alfa (INN) and agalsidase beta (INN). This enzyme is a homodimeric glycoprotein that hydrolyses the terminal alpha-galactosyl moieties from glycolipids and glycoproteins. It predominantly hydrolyzes ceramide trihexoside, and it can catalyze the hydrolysis of melibiose into galactose and glucose.
Storage:	Shipped and store at 4 °C for 6 months.
Synonyms:	Alpha-Galactosidase Kit; Alpha-Galactosidase Assay; Alpha-Galactosidase; EC 3.2.1.22; Melibiase; GLA; GALA; 9025-35-8
Size:	100 Assays
Kit Components:	96-Well Microplate: 1 plate Assay Buffer: 30 ml x 4; 4 °C Reaction Buffer: 4 ml x 1; 4 °C Substrate: Powder x 2; -20 °C Stop Solution: 15 ml x 1; 4 °C Standard (1 mmol/L): 1 ml x 1; 4 °C Plate Adhesive Strips: 3 Strips Note: Substrate: For each tube, add 1 ml distilled water to dissolve before use.
Materials Required but Not Supplied:	1. Microplate reader to read absorbance at 400 nm 2. Distilled water 3. Pipettor 4. Pipette tips 5. Mortar 6. Centrifuge 7. Timer 8. Ice
Target Species:	Detection and Quantification of Alpha-Galactosidase (α-GAL) Activity in Tissue extracts, Cell lysate, Cell culture media and Other biological fluids Samples.
Preparation:	1. For cell and bacteria samples Collect cell or bacteria into centrifuge tube, discard the supernatant after centrifugation, add 1 ml Assay buffer for 5 × 10^6 cell or bacteria, sonicate (with power 20%, sonication 3s, intervation 10s, repeat 30 times); centrifuged at 10,000g 4 °C for 20 minutes, take the supernatant into a new centrifuge tube and keep it on ice for detection. 2. For tissue samples Weigh out 0.1 g tissue, homogenize with 1 ml Assay buffer on ice, centrifuged at 10,000g 4 °C for 20 minutes, take the supernatant into a new centrifuge tube and keep it on ice for detection.
Assay Protocol:	Add following reagents into the microplate: Reagent Sample Control Standard Blank Sample 10 μl 10 μl -- -- Distilled water -- 20 μl -- -- Substrate 20 μl -- -- -- Reaction Buffer 40 μl 40 μl -- -- Mix, put it in the oven, 37 °C for 30 minutes. Stop Solution 130 μl 130 μl -- 200 μl Standard -- -- 200 μl -- Mix, record absorbance measured at 400 nm.
Analysis:	Unit Definition: One unit of α-Galactosidase activity is the enzyme that generates 1 μmol of p-nitrophenol per hour. 1. According to the protein concentration of sample α-GAL (U/mg) = Cstandard × (OD Sample - OD Control) / (OD Standard - OD Blank) / (Cprotein ×Vsample) / T = 200 × (OD Sample - OD Control) / (OD Standard - OD Blank) / Cprotein 2. According to the weight of sample α-GAL (U/g) = Cstandard × (OD Sample - OD Control) / (OD Standard - OD Blank) × Vtotal / (Vsample × W/ Vassay) / T = 40 × (OD Sample - OD Control) / (OD Standard - OD Blank) / W 3. According to the quantity of cells or bacteria α-GAL (U/10^4) = Cstandard × (OD Sample - OD Control) / (OD Standard - OD Blank) × Vtotal / (Vsample × N/ Vassay) / T = 40 × (OD Sample - OD Control) / (OD Standard - OD Blank) / N Cprotein: the protein concentration, mg/ml; Cstandard: the concentration of Standard, 1 mmol/L = 1 μmol/ml; W: the weight of sample, g; N: the quantity of cell or bacteria, N × 10^4; Vtotal: the total volume of the enzymatic reaction, 0.2 ml; Vsample: the volume of sample, 0.01 ml; Vassay: the volume of Assay buffer, 1 ml; T: the reaction time, 0.5 hour;

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