b-Galactosidase Activity Assay -- Marian Price-Carter, 9/7/00
Day 1: Start overnight cultures in assay medium.
Negative control: cells lacking b-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, ONPG2.
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 times.
Vortex; equilibrate the tubes 5' in a 28 C water bath.
Start reaction by adding 0.2 mL substrate, o-nitrophenyl-b-D-galactoside (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 b-galactosidase enzyme activity.
- 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 Na2CO34.
- Vortex. - Note time of addition precisely.
- 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 activity6.
b-Galactosidase is able to hydrolyze (cleave) b-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-b-D-galactopyraniside (ONPG) is used in place of lactose. When the b-galactosidase cleaves ONPG, o-nitrophenol is released. This compound has a yellow color, and absorbs 420 nm light. To measure b-galactosidase activity the accumulation of yellow color (increase 420 nm absorbance)/minute is monitored.
1 In Salmonella (which is naturally b-galactosidase minus) this assay is used to monitor transcription from insertion elements (that encode the b-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 b-galactosidase assays
Z buffer, per 50 mL:
Note: BME is added to the reaction buffer to stabilize the b-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 MgSO4 (0.001M)
- 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 yellow.
- 1.61g Na2HPO4.7H2O (0.06M)
- 0.55g NaH2PO4.H2O (0.04M)
- adjust the pH to 7.0
- phosphate buffer is stable at room temperature and does not need to be made fresh each time.
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 even further!
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:
light scattering 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 OD600) Typical values:
The units give the change in A420/min/mL of cells/OD600
- OD420 and OD550 are read from the reaction mixture.
- OD600 reflects cell density in the washed cell suspension.
- T = time of the reaction in minutes.
- V = volume of culture used in the assay in mLs.
a fully induced lac+ operon (+IPTG) = 1500 units
an uniduced lac+ operon (no IPTG) = 1.5-3 units