|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 22.214.171.124; Melibiase; GLA; GALA; 9025-35-8|
|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
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
4. Pipette tips
|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;