b-Galactosidase Assay with CPRG
- Lyse cells
(recommended cell lysis buffer: 0.25M Tris/HCl pH 8.0, 0.25% (v/v) NP40,
2.5 mM EDTA)
- Pipet about 10 µl of extract into a 96-well plate (appropriate
neg. control / blank: 10 µl of mock transfected cells, or
non-transfected cells – as there is a slight endogenous b-Gal
activity) – leave one well empty for blank (A-1)
- Add 100 µl substrate solution
to the wells (also to the blank-well)
- Incubate until red color develops
(min to hours – depending on b-Gal activity, if you
have low activity you can also incubate at 37°C)
- Optional: Stop with 50 µl of
Stop solution (only necessary if you want to time it exactly, e.g. by
adding the substrate in a timed way and stopping the reaction in the same
way)
- Measure with ELISA Reader at 570 nm (Filter #3)
Lysis Buffer:
0.25M Tris/HCl pH 7.4 (or better 8.0)
0.25% (v/v) NP40
2.5 mM EDTA
CPRG-substrate
solution: 1 mg/ml (= 1.65 mM)
in PBS + 10 mM KCl, + 1 mM MgCl2
alternative substrate buffer:
60 mM Na2HPO4
pH 8.0, 1 mM MgCl2, 10 mM KCl, 50 mM Mercapto-ethanol
Stop solution: 0.5M Na2CO3