Determination of β-galactosidase in cellular extracts of experimental
This protocol describes the determination of expressed β-galactosidase activity after transfection of mammalian cells with the reported molecular vector (see Figure 17-5). This method is both simple and rapid, and can be measured using a visible spectrophotometer. This experiment was derived from the next volume of the Laboratory Guide to Molecular Cloning (3rd edition) by [American] J. Sambrook D.W. Russell.
Operation method
Experimental determination of beta-galactosidase in cellular extracts
Materials and Instruments
Cultured mammalian cells transfected with the DNA of interest Move makings For more product details, please visit Aladdin Scientific website.
E. coli β-galactosidase Tris-Cl sodium phosphate ONPG Na2CO3 β-mercaptoethanol MgCl2
Buffers and solutions
Dilute the storage solution to the appropriate concentration.
100XMgCl2
0.1mol/L MgCl2
4.5mo1/L β-mercaptoethanol
Before use, add an appropriate amount of 14.7 mol/L β-mercaptoethanol storage solution.
Na2CO3 (1mol/L)
10.6 g anhydrous solid was dissolved in 100 ml of water.
1xONPG
ONPG was dissolved in 0.1 mol/L sodium phosphate (pH 7.5) at a concentration of 4 mg/ml.
Sodium phosphate (0.1 mol/L, pH 7.5)
Mix 41 ml of 0.2mol/L Na2HPO4-2H2O ( Mt=178.05; 35.61 g/L), 9 ml of 0.2mol/L NaH2PO4-2H2O ( Mr=156.01; 31.21 g/L) and 50 ml of water.
Tris-Cl (1 mol/L, pH 7.8). 
Enzymes and buffers
Escherichia coli β-galactosidase
This enzyme is commercialized (e.g. Sigma)
Additional reagents
Step 1 of this protocol requires the reagents listed in Protocol 5 of this chapter.
Cells and Tissues
Cultured mammalian cells transfected with the DNA of interest
Using one of the transfection protocols in Chapter 16, transfect cells with a plasmid bearing the galactosidase reporter gene (e.g., CLONTECH's reporter molecule series vectors; see Figure 17-5). 
Methods
1. Prepare a cell extract from the transfected cells as described in steps 1-4 of protocol 5. Approximately 30ul of the extract is taken for the β-galactosidase assay. The exact amount of extract required is determined by the strength of the promoter driving β-galactosidase gene expression, the transfection efficiency, and the incubation time for the assay. If heat treatment is to be used to inactivate endogenous β-galactosidase, incubate the cell lysates at 50°C for 45-60 min prior to the assay. preheating will also inactivate luciferase, so if preheating is used, the luciferase and β-galactosidase activity assays should be performed separately with separate portions of cell lysate.
2. For each sample of transfected cell lysate used for the assay, mix:
100xMg2+ solution 3ul
1xONPG 66ul
Cell extract 30ul
0.1mol/L sodium phosphate (PH7.5) 201ul
Negative and positive controls are required to check for the presence of endogenous inhibitors and β-galactosidase, respectively. All control experiments should contain 30ul of lysate from pseudotransfected cells. In addition, positive controls should include 1ul of commercial E. coli β-galactosidase product <50 units/ml. the commercial enzyme product should be dissolved in 0.1mol/L sodium phosphate (PH7.5) at a concentration of 3000 units/ml. 1ul of β-galactosidase reservoir solution should be added to 60ul of 0.1mol/L sodium phosphate (PH7.5) before use. Before use, 1ul of β-galactosidase storage solution was added to 60ul of 0.1mol/L sodium phosphate (PH7.5) to make 50 units/ml of enzyme working solution. Each unit of E. coli galactosidase is defined as the amount of enzyme used to hydrolyze 1umol of ONPG substrate at 37°C for 1 min.
3. Incubate the reaction solution at 37°C for 30 min or until a pale yellow color appears. The background of endogenous β-galactosidase is very low in most cell types, allowing for incubation times as long as 4 to 6 h. The reaction is incubated at 37°C for 30 min or until a yellow color appears.
4. Terminate the reaction by adding 500ul of 1mol/L Na2CO3 to each tube. Read the optical density value at 420 nm on a spectrophotometer.
The linear range of measurement is 0.2 to 0.8 OD420. If the measurement is outside the linear range, repeat the experiment with a smaller amount of protein extract. The extract can be diluted with 0.25 mol/L Tris-Cl (pH 7.8) to reduce the protein concentration. 