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.

Reference: Simon and Lis (1987) NAR 15: 2971-2988.