b-galactosidase Solution Assay

 

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 MgCl ₂

up to 100 ml with Q

store at room temperature

1M KPO₄ pH 7.5

250 ml 1M KH₂ PO₄ (monobasic) 68.05 g up to 500 ml

100 ml 1M K₂ HPO₄ (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 Na₂ CO₃ 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.