b-galactosidase Solution Assay
Reference: Simon and Lis (1987) NAR 15: 2971-2988.
Solutions
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°
Procedure
• 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.
