Beta-galactosidase: Beta-galactosidase is encoded by the E. coli lacZ gene and catalyzes the hydrolysis of galactosides. The biggest advantage is the ease of observation of in situ expression by immunohistochemistry, which is one of the most commonly used reporter genes for monitoring transfection rates. Enzyme activity can be detected by standard colorimetric method using o-nitrobenzene-β-D-galactopyranoside (ONPG) as a substrate, and the detection kinetics range is 6 orders of magnitude. Chlorophenol red-β-D-galactopyranoside (CPRG) is another substrate that can be used to detect enzyme activity by colorimetry, and its sensitivity is nearly 10 times higher than that of ONPG. Using MUG and fluorescein digalactoside (FDG) as substrates, the activity can be detected by fluorescence. This method detects the enzymatic activity of individual cells and can be used for flow cytometry (FACS) analysis. For example, dioxetane can be used as a substrate to detect enzymatic activity by chemiluminescence. The detection range is the largest and the sensitivity is the highest, which is similar to the sensitivity of detecting luciferase activity by bioluminescence. Reagents and equipment: experimental method:
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1. Reaction buffer: 100 mmol / L sodium acetate, 100 mmol / L KCl, 2% (volume fraction) Triton X-100, 5 mmol / L dithiothreitol (DTT), pH 4.3;
2. Substrate: 1.7mmol/L 4-methyl-umbelliferone-β-D-galactoside (4-MUG) or o-nitrobenzene-β-D-galactopyranoside (ONPG), dissolved In the reaction buffer;
3. Microcentrifuge;
4. Fluorometer: the transmitted light wavelength is 355nm, the excitation light wavelength is 460nm, and the cuvette can hold 200μl sample;
5. Flow cytometry (FACS) analysis
6. 37 ° C water bath;
Mammalian primary cell culture.
Primary cells are collected.
1. Dilute with twice the volume of buffer, then centrifuge in a microcentrifuge for 10 min at 0-4 ° C to obtain a precipitate with a gradient;
2. Resuspend the pellet in 0.25 ml of reaction buffer;
3. Mix with an equal volume of substrate and incubate for 1 h at 37 °C. Use 0.25 ml of reaction buffer instead of substrate as a sample blank;
4. There is a blank for the substrate;
5. Fluorescence was measured after 1 h;
6. Flow cytometry (FACS) analysis
Î’-galactosidase (fluorescence assay) in mammalian primary cell culture
Î’-galactosidase (fluorescence assay) in mammalian primary cell culture