Î’-galactosidase (spectrophotometry) of mammalian primary cell culture
Î’-galactosidase (spectrophotometry) of mammalian primary cell culture
Reagents and equipment:
1. Reaction buffer: 5 g / L polyethylene glycol octyl phenyl ether (Triton X-100), 0.05 mol / L sodium citrate-phosphate buffer, pH 4.3;
2. Stop solution: 0.25% glycine-NaOH, pH 10;
3. Substrate: 6mmol / L p-nitrophenyl-β-galactofuranoside dissolved in a buffer solution;
4. Microcentrifuge with 1.5-2.0ml small tube;
5. Spectrophotometer (visible wavelength) with 1 ml cuvette;
UV-visible spectrophotometric determination of enzyme activity: The activity of β-galactosidase was determined using ONPG as a substrate.
Enzyme activity definition: using ONPG as a substrate, enzyme hydrolysis at 37 ° C, the amount of enzyme releasing lμmol / L o-nitrophenol per minute, defined as one enzyme activity unit.
experimental method:
1. For each test, mix 0.5 ml of the substrate with a suspension of the membrane fraction or 50 μl of membrane precipitated in a microcentrifuge tube;
2. If the empty cup is set in step 1, immediately add 1 ml of stop (buffer);
3. The incubation solution was incubated at 37 ° C for 30 min;
4. Add 1 ml of the stop (buffer) to the test solution;
5. Centrifuge in a microcentrifuge for 1-2 minutes to remove any precipitated material;
6. Read the absorbance of the liquid phase for the blank at a wavelength of 410 nm;
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