β-Galactosidase Activity Assay -- Marian
Day 1: Start overnight cultures in assay medium.
Negative control: cells lacking β-galactosidase, such as LT2; positive
control: cells with high enzyme activity.
Day 2: Dilute cells 1/100 in fresh medium, grow to
mid-log.1 Prepare solutions: Z buffer, phosphate buffer,
Preparation of Cells
Incubate cultures 20' on ice to stop growth and wash:
Dilute cells in Z buffer to 1 mL (most easily done
with a pippeter). For most activities, 0.5 mL cells + 0.5 mL Z buffer
will produce a desirable amount of yellow color in 1-2 hours. For
higher levels (>500 Miller units), try 0.1 mL cells +
0.9 mL Z buffer.
- Pellet at least 2 mL of cells at 4 C by
centrifuging 10' at 6,000 rpm in a Sorval SS34 rotor.
- Pour off the supernatant.
- Resuspend the cell pellet in the same volume
of chilled Z buffer.
- Measure the OD600 of the
resuspended cells (blank against Z buffer)
Permeabilize the diluted cells by adding 100 µl chloroform
and 50 µl 0.1% SDS (sodium dodecyl sulfate, sodium laurel
sulfate). Chloroform is easier to pippete if the air in the pippete
tip is saturated by drawing up and releasing chloroform several
Vortex; equilibrate the tubes 5' in a 28 C water bath.
Start reaction by adding 0.2 mL substrate,
(ONPG; 4 mg/mL)
This is basically the assay described by J.H.
Miller in "Experiments in Molecular Genetics" 1972 Cold Spring Harbor
Laboratories pages 352-355, with an extra step added. In the assay
described here, the cells are pelleted and resuspended in assay buffer
(Z buffer) to eliminate error due to the effects of different carbon
sources in the growth medium on the β-galactosidase enzyme
- Vortex - Record the time of addition
precisely with timer or stopwatch.
- Incubate the cells at 28 C.
- Stop the reaction after sufficient yellow
color has developed3 by adding 0.5 mL 1M
- Vortex. - Note time of addition
- Transfer 1 mL to an eppendorf tube, spin 5'
at maximum to remove debris and chloroform.
- Record the optical density at 420 nm and at
550 nm for each tube. 5
- Calculate the units of
β-Galactosidase is able to hydrolyze
(cleave) β-D-galactosides. This enzyme facilitates growth on
carbon sources like lactose by cleaving it into a molecule of glucose
and a molecule of galactose which the cells can catabolize and grow
on. In the assay described above, the substrate
o-nitrophenyl-β-D-galactopyraniside (ONPG) is used in place of
lactose. When the β-galactosidase cleaves ONPG, o-nitrophenol is
released. This compound has a yellow color, and absorbs 420 nm
light. To measure β-galactosidase activity the accumulation of
yellow color (increase 420 nm absorbance)/minute is monitored.
1 In Salmonella (which is naturally
β-galactosidase minus) this assay is used to monitor
transcription from insertion elements (that encode the
β-galactosidase enzyme) that have inserted into different
genes. The assay is usually performed on cells in the mid-log phase of
growth. On rich carbon sources like glucose, the OD600 of a
culture of wild-type Salmonella in mid-log phase ranges from
0.28-0.7. On poorer carbon sources or in strains that have mutations
in genes that are important for growth, the OD600 at
mid-log phase may be lower, since the cells may enter stationary phase
at a lower density. Therefore, before doing the assay, it is important
to follow the growth of the strain of interest in each type of medium
that will be used, plot a growth curve, and determine when the cells
are in mid-log phase in that particular medium.
2 Solutions for β-galactosidase assays
Z buffer, per 50 mL:
Note: BME is added to the reaction buffer to
stabilize the β-galactosidase enzyme. The important part of BME
is a reactive thiol (SH group). Thiols react with oxygen in the air
and oxidize (inactivate) over time. Therefore, try not to make much
more Z buffer than you will use in a few days. Store the unused
portion at 4 C.
- O.80g Na2HPO4.7H2O (0.06M)
- 0.28g NaH2PO4.H2O (0.04M)
- 0.5 mL 1M KCl (0.01M)
- 0.05 mL 1M
- 0.135 mL b
-mercaptoethanol (BME) (0.05M)
- bring to approximately 40 mL with
H2O, dissolve all the salts
- adjust the pH to 7.0
- use a graduated cylinder to bring the buffer
to 50 mL
- store at 4 C.
ONPG should be dissolved fresh each day. Dissolve 1.5X as much as
you think you will need, because you may have to repeat one or more of
the assays i.e. for a different amount of time or with a
different cell dilution. Dissolve the ONPG to a final concentration of
4mg/mL in 0.1M phosphate buffer pH 7.0.
Phosphate buffer, per 100 mL:
3 What is sufficient yellow
color? To get the most accurate measure of activity, the absorbance at
420 nm (A420) should range from 0.6 to 0.9. Readings as low
as 0.1 and as high as 1.2 are acceptable. Tubes that have become as
yellow as a tube of (unused) LB broth will probably be sufficiently
- adjust the pH to 7.0
- phosphate buffer is stable at room
temperature and does not need to be made fresh each
If the reading is too low, try the assay again with more cells or
longer incubation time. When the element has inserted into a gene that
is not expressed much, it will probably take hours to develop enough
yellow color. If your negative control starts to turn yellow (after
several or more hours) it means that the substrate is beginning to
auto-hydrolyze. The assay can be left overnight. The auto-hydrolysis
is then accounted for by subtracting the A420 and
A500 of the negative control from that of the tests before
doing any further calculations.
If the reading is too high, try the assay again with fewer cells. Aim
to stop the reaction after 15 minutes. For example, if in your first
attempt, you added 0.5 mL of cells + 0.5 mL of Z buffer, and it was
too yellow after 5 minutes, try adding 0.1 mL cells + 0.9 mL of Z
buffer. Watch the tube carefully. Some cultures may have to be diluted
4 Adding the 1 M Na2CO3
stops the reaction by raising the pH of the solution to 11. At this
pH the enzyme is not active.
5 The reading at 420 nm is a combination of
absorbance by o-nitrophenol and light scattering by cell debris. The
absorbance at 550 corrects for light scattering. There is no
absorbance from o-nitrophenol at this wavelength. The light scattering
at 420 nm is proportional to that at 550 nm:
at 420 nm = 1.75 x OD550
6 Use the following equation
to calculate units of enzyme activity:
Miller Units =
1000 x [(OD420 - 1.75 x OD550)] / (T x V x
The units give the change in A420/min/mL of
- OD420 and OD550 are
read from the reaction mixture.
- OD600 reflects cell density in
the washed cell suspension.
- T = time of the reaction in
- V = volume of culture used in the assay in
a fully induced
lac+ operon (+IPTG) = 1500 units
an uniduced lac+ operon (no IPTG) = 1.5-3 units