Bio-Rad Fluorescent Assay Kits User Manual
Page 5
MUG and 42.3 µl of 14.2 M ß-mercaptoethanol to 50 ml of 1x
Reaction Buffer.
3. Add 500 µl assay buffer prepared above to each 1.5 ml microfuge
tube.
4.
Add 1.5 µl of distilled water to the negative control tube.
5.
Add 1.5 µl of purified ß-galactosidase (supplied with the kit) to the
positive control tube.
6.
Add sample. As a guideline, 2–4 µg of total protein should fall within
the linear range of 4MU detection.
7. Incubate reactions in a 37 °C water bath for 15 minutes.
8. While samples are incubating, label fluorometer cuvettes and add 1.0 ml
1x Stop Buffer to the cuvettes.
9.
Stop the reactions by pipetting the entire reaction volume into the
cuvettes containing 1.0 ml 1x Stop Buffer. Pipet samples up and
down to mix.
10. Read samples as soon as possible; prolonged incubation may lead to
a decrease in fluorescence signal.
4.5 Reading Samples
A. Standard Curve
Note: Use cuvettes prepared in Section 4.3.
1.
Zero the VersaFluor by placing the cuvette containing 1x Stop
Buffer (cuvette 5) in the cuvette holder. Close sample compartment
lid. Wait approximately 2–3 seconds for the detector to stabilize.
Press the SET ZERO button.
2.
Insert cuvette 1, highest concentration cuvette, into the cuvette holder.
Close the sample compartment lid. Wait approximately 2–3 seconds
for the detector to stabilize.
3.
Set the range to 14,000. Remove cuvette.
Note: Do not change range settings after this point.
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4.3 Standard Curve Preparation
Generate a calibration curve of 4-methylumbelliferone (4MU) from
10 nM to 10,000 nM in 1x Stop Buffer. Add 1x Stop Buffer and 4MU to
each cuvette as described in the table below. Mix the solutions using a
new disposable pipet for each cuvette or by placing parafilm on the top of
the cuvette and inverting a few times.
Cuvette
4MU
1x Stop Buffer (ml)
[4MU] (nM)
1
20 µl of 1 mM 4MU stock
1.98
10000
2
200 µl of cuvette 1
1.80
1000
3
200 µl of cuvette 2
1.80
100
4
200 µl of cuvette 3
1.80
10
5
0
2.00
0
4.4 ß-galactosidase Assay
Cell extracts prepared by the traditional freeze-thaw method
2
or with
the usage of a nonionic detergent, 0.1% Triton X-100 or Nonidet-P40
1,3
may be assayed. In addition to the negative (no enzyme) and positive
(purified ß-galactosidase) controls, it is recommended that a mock cell
lysate (non-transformed preparation) be included in the assay to detect
endogenous ß-galactosidase activity.
Note: For accurate results, samples must be kept on ice until all enzyme
mixtures have been added.
1.
Keep ß-galactosidase and ß-mercaptoethanol on ice. Thaw 4MU and
MUG at room temperature. Do not thaw at elevated temperatures as
this may cause precipitation. Vortex 4MU and MUG into solution.
2.
Prepare fresh assay buffer by diluting 100 mM MUG and 14.2 M
ß-mercaptoethanol to a final concentration of 0.6 mM MUG and
12.0 mM ß-mercaptoethanol in 1x Reaction Buffer. Vortex to mix.
Example: 100 samples would require 50 ml of assay buffer (500 µl/
sample). To prepare 50 ml of assay buffer, add 300 µl of 100 mM
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