-Galactosidase assays
1. Thaw the system components and mix each component well before use.
Place the Assay 2X Buffer on ice.
2. Pipet 150 l of the diluted cell extracts (100 l of cell extracts + 50 l of 1X Reporter
Lysis Buffer) into labeled tubes
3. Add 150 l of Assay 2X Buffer to each of the tubes.
4. Mix all samples by vortexing briefly.
5. Incubate the reactions at 37 C for 30 min. [Color development continues for 3 h]
6. Stop the reactions by adding 500 l of 1 M Sodium Carbonate. Mix by vorexing
briefly.
7. Read the absorbance at 420 nm.
* Standard Curves
-Galactosidase
Volume of
Volume of 1X
Standard (milliunits)
1:10,000 Stock (l)
Reporter Lysis Buffer (l)
0
0
150
1.0
10
140
2.0
20
130
3.0
30
120
4.0
40
110

50
100

60
90
1:10,000 Stock : Add 10 l of 1u/l -galactosidase to 990 l of 1X Reporter
Lysis Buffer and mix. Then add 10 l of this 1:100 dilution to 990 l of 1X
Reporter Lysis Buffer and mix it to make a 1:10,000 stock solution.
(1) Follow the protocol described above, Steps 3-7.
(2) Plot the absorbance at 420 nm versus concentration of –Galactosidase standards.
1
Dr. Lee’s Lab