-GALACOTSIDASE ASSAY
This assay is based on the hydrolysis of O-Nitrophenyl -d galactoside (ONPG). ONPG is colorless,
but when the enzyme breaks the -galactoside bond (between ONP and galactose), the released ONP
group absorbs in the visible range (420 nm) and appears yellow.
Enzyme activity is expressed in “Units”, and in this assay, a unit is defined as the amount of enzyme
that will hydrolyze 10-9 moles of ONPG in 1 minute at 37 C. Many parameters of this assay have
been established so that the absorbance is proportional to the amount of enzyme present, and the
results are easy to convert into Units.
Reagents needed for -galactosidase assay:
Z buffer (See below)
0.1 M sodium phosphate buffer, pH 7.5
1 M Na2CO3
ONPG (o-nitrophenyl -d-galactoside), 4 mg/mL in 0.1 M sodium phosphate buffer, pH 7.5
It is best to make this fresh. When it turns yellow, discard it and make more
Beware: we have both ONP--d-galactoside and ONP--d-galactoside. -gal will not cleave
the -galactoside bond!
Protocol: NOTE: it is strongly recommended that the members of your group do this together.
This is written for 1 mL volumes, to be carried out in small test tubes and then transferred to plastic
cuvettes for reading. It is possible to do the assay in larger tubes that can be read directly in the Spec
20 – double the volumes [in brackets].
1. Add 10-50 uL of sample to 1 mL of Z buffer and equilibrate to 37 C [20 uL in 2 mL of Z buffer]
2. Prepare a blank tube including the same volume of the same buffer the sample is in, add 1 mL of
Z buffer [2 mL], and equilibrate it to 37 C.
3. Start the reaction by adding 0.1 mL [0.2 mL] of ONPG solution to each tube, and mix (vortex)
4. Incubate the samples at 37 C for at least 10 minutes*, and until a faint yellow color appears (some
samples may take significantly longer than 10 minutes)
They need not be all the same amount of time, but that does make your assay easier, both to do and to
calculate. Also you should have a time-matched blank.
5. Stop the reaction by adding 0.5 mL [or 1.0 mL] of 1 M Na2CO3and record the length of time for
each sample.
6. Blank the spectrophotometer using the control prepared in step 2, and read the samples
The enzyme activity of the -galcotosidase is defined in units as
A420 X 380 [760]
= Units of -galactosidase
Min at 37 C
Finally To express this as a concentration of enzyme Units/mL, divide by the # of mL used in the
assay AND remember to multiply by any dilution factor you used!!
What is the Factor of 380??
The factor of 380 takes into account the molar concentration of the ONPG and the extinction
coefficient of the product, ONP.
Make sure you are using the readable range of the assay: A420 of 0.1 to 0.8.
This assay is set up to work in a nearly linear fashion in a particular range. Obviously, if all of the
substrate is converted by the time you stop the reaction, there is no way to tell how long that took, and
since you measure the enzyme in terms of moles converted per unit time, your data would be
meaningless. A more subtle problem is that the interpretation of substrate conversion to enzyme
amount depends on the enzyme operating at a constant velocity. The only way to ensure this is to
keep the substrate concentration high where the velocity is maximum (V max). Under such conditions,,
only a small fraction of the substrate is converted during the time of the assay, so [S] does not decrease
significantly (because that would decrease the velocity). If [S] decreases too much, as the reaction will
slow, the Units (which are substrate converted in a given time) will be lower than they should be, so
you will underestimate the amount of enzyme you have. Aim for an abosrbance between 0.1 and 0.8,
achieved in 2 minutes or more. You can make yourself some “eyeball” standards, so that you let your
reactions go long enough, but not too long, before stopping them
It might be reasoned that you could just assay for a much shorter time, but then you run into problems
of precision in the timing of start and stop, which will affect your assay adversely. The protocol
suggests that you let the reactions go at least 10 minutes. I think you can probably go with somewhat
shorter time, such as 2 to 5 minutes, but don’t try to get away with assays under two minutes. If
they are heading toward completion that fast, start over with less of (or a more dilution version of) the
sample.
On the other hand, keep in mind that if you do not see any conversion in your standard amount of
time, that does not mean there is no enzyme there! You can let the reaction go quite a bit longer (as
long as you have a control tube going just as long, to control for enzyme-independent substrate
conversion). Very low absorbances are often too imprecisely read by the spectrophotometer to be
believed. If they are smaller than the standard deviation of the assay (or the ability of the
spectrophotometer to read them) then they are basically indistinguishable from zero. One of the
advantages of this assay is that you can add up to 50 uL of extract, and carry the reaction out to 30
minutes. If, after using maximal conditions, you still get a very low absorbance, you could say, “ the
concentration of enzyme in this fraction is less than ____”, rather than claim there is no enzyme there
at all. (Exception: when reading the column fractions, it is not necessary to try to get a good reading
on fractions that contain very small amounts of the enzyme)
Z Buffer
4.26 g
2.4 0g
.375 g
0.06 g
5 mM
I have two recipes for this. Either will work– are they the same??
Na2HPO4 (anhydrous)
NaH2PO4 (anhydrous)
KCl
MgSO4
DTT *
adjust to pH 7
q.s. 500 mL
1.61 g Na2HPO4 7H2O
5.50 g NaH2PO4 H2O
0.75 g KCl
0.25 g MgSO4 7H20
2.7 mL 2-mercaptoethanol
adjust the pH to 7 and
bring to 1 L, do not autoclave