-galactosidase assay

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-galactosidase assay

-galactosidase assay

The purpose of this experiment is to measure the relative transcription of a Xgal+ MudJ insertion
mutant and regulatory mutants. Since the MudJ operon fusions express the lacZ gene product (galactosidase;)
from the promoter of the mutated gene, transcription of the mutant gene can be quantitated
by determining the -galactosidase activity expressed in the MudJ insertion mutants (Beckwith, 1981). By
determining the -galactosidase activity expressed in regulatory mutants or under different growth
conditions, the transcriptional regulation of the mutant gene can be studied. -galactosidase assay.
-galactosidase can be assayed by measuring hydrolysis of the chromogenic substrate, o-nitrophenyl--Dgalactoside
(ONPG).

The amount of o-nitrophenol formed can be measured by determining the absorbance at 420 nm. If excess
ONPG is added, the amount of o-nitrophenol produced is proportional to the amount of -galactosidase
and the time of the reaction. The reaction is stopped by adding Na2CO3 which shifts the reaction mixture
to pH 11. At this pH most of the o-nitrophenol is converted to the yellow colored anionic form and galactosidase
is inactivated. The reaction can be run using whole cells that have been permeabilized to
allow ONPG to enter the cytoplasm. However, since whole cells are present, the absorbance at 420 nm is
the sum of the absorbance due to o-nitrophenol and light scattering due to the cells. The contribution of
light scattering can be determined by measuring the absorbance at 550 nm where o-nitrophenol doesn't
absorb. The light scattering at 420 nm is 1.75x the light scattering at 550 nm, so the absorbance of onitrophenol
is determined by subtracting 1.75 x OD550 nm. The corrected absorbance is then used to
calculate the activity of -galactosidase.

REFERENCE
Miller, J. 1972. Experiments in Molecular Genetics, p. 352-355. Cold Spring Harbor Laboratory, NY.

-galactosidase assay:
1. Subculture each strain in 2 ml of the appropriate medium (plus any required supplements). Grow
overnight at 37°C.
2. Add 1.5 ml of the culture to a sterile microfuge tube. Spin for 20 sec in the microfuge. Pour off the
supernatant and resuspend the cell pellet in 1 ml 0.85% NaCl. Vortex until completely resuspended.
3. Subculture each cell suspension into 5 ml medium in a Klett flask. (Try to start each culture at about
10 KU.) Grow at 37°C to exponential phase (100-120 KU).
4. Prepare triplicate dilutions of 0.5 ml cells with 0.5 ml complete Z-buffer in test tubes. Also prepare
two controls with only 1.0 ml Z-buffer. (Save the cell suspension on ice!)
5. To permeabilize cells add 1 drop of 0.1% SDS and 2 drops of chloroform using a Pasteur pipet.
Vortex.
6. Place the tubes in a 30°C water bath and allow to equilibrate for about 2 min.
7. Add 0.2 ml ONPG to each tube and vortex to initiate the reaction. Return to the 30°C shaker. Note the
time.
8. When a yellow color develops, stop the reaction by adding 0.5 ml of 1 M Na2CO3. Note the time that
each reaction is stopped.
9. Return to the shaker for about 5 min.
10. Determine the absorbance within 1 hr. Measure OD420 and OD550 for each tube.
11. Measure OD650 of the cell suspension. If the OD650 is greater than 1.2, dilute the cells (0.5 ml cells +
0.5 ml 0.85% NaCl) and reread the absorbance. If the cells are diluted, remember to correct the OD by
the dilution factor before calculating -galactosidase activity.
13. Using the equation shown below, calculate the -galactosidase activity of each sample. Determine the
mean and standard deviation of the triplicates.

Activity = OD420 - (1.75 OD550) x 1 nmol x l.7 ml
------------------------- -----------
OD650 x time x vol 0.0045 ml cm

Where:
time = time of reaction in min
vol = ml cells added to the assay tubes
0.0045 OD420/nmol = e420 o-nitrophenol
1.7 ml = total vol
cuvette = 1 cm path length
Activity = nmol / min / OD650 ml

REAGENTS
Z-buffer stock solution
4.27 g Na2HPO4
2.75 g NaH2PO4 H2O
0.375 g KCl
0.125 g MgSO4 7H2O
Adjust to pH 7.0.
Bring to 500 ml with dH2O. Do not autoclave. Store at 4°C.

For complete Z-buffer -- Prior to daily use mix:
50 ml Z-buffer
0.14 ml -mercaptoethanol

ONPG (4 mg/ml) (Sufficient for 100 assays -- make fresh daily)
80 mg o-nitrophenyl--D-galactoside (o-nitrophenyl--D-galactopyranoside)
20 ml dH2O

1 M Na2CO3 (Sufficient for 100 assays -- store in refrigerator)
5.3 g Na2CO3
50 ml dH2O

Link: http://www.sci.sdsu.edu/~smaloy/Research/pdf%20files/B-galactosidase.pdf