b-galactosidase Solution Assay
Reference: Simon and Lis (1987) NAR 15: 2971-2988.
CPRG Assay Buffer
50 mM KPO4 pH 7.5 5 ml 1M KPO4 pH 7.5
1 mM MgCl2 0.1 ml 1M MgCl2
up to 100 ml with Q
store at room temperature
1M KPO4 pH 7.5
250 ml 1M KH2PO4 (monobasic) 68.05 g up to 500 ml
100 ml 1M K2HPO4 (dibasic) 87.1 g up to 500 ml
50 mM CPRG
0.0304 g chlorophenyl red b-galactopyranoside
(Boehringer Mannheim #834-308)
up to 1 ml with Q
store in the dark at -20°
Wash cells or tissue twice in PBS, pellet and resuspend in 150 microliters of 0.25 M Tris HCl pH 8.0.
Freeze thaw the cells 3 times by alternating between dry ice and 37°.
Spin for 15 minutes at 4° and transfer the supernatant to a new tube and hold on ice.
Mix the following reaction solution per sample:
264 microliters CPRG Buffer
6 microliters (50mM CPRG)
30 microliters of the aforementioned cell extract
Incubate at 37° until a deep orange color appears.
Add 500 microliters of 1 M Na2CO3 to terminate the reaction.
Immediately read the OD574 and keep in mind that the linear range is between A574 of 0.4 and 2.7.
To compare between samples, normalize the time, volume of lysate and protein concentration.