eek 5 - Phosphatase & Invertase Activities
W
Danny VanDuzer, Misong Sim, and Nicky Faustini
rotocol
P
Part 1 - Phosphatase Activity Assay
1. Make the positive control by diluting 0.500 mM PNP to 0.100 mM in a volume of 700
through the combination of140 μL of 0.500 mM PNPwith 560 μl of 10.0 mM Tris pH
9.0.
2. The number of samples determined to be tested is 17 which includes a negative and a
positive control.
3. To test each sample, add a diluted concentration of 1.0mM PNPP; use 200 μl as your
final volume. Once you calculate the volume of 10.0 mM PNPP needed for one sample,
multiply it by the total number of samples.
4. Dilute the PNPP with 10.0 mM Tris at a pH of 9 using 20 μl PNPP and 170 μl Tris. Note
that these values have been multiplied by 1.2 in order to account for any transfer error
whilst pipetting.
a. Therefore, – considered using 20 μL of PNPP – in one sample, 170 μL of 10.0
mM PNPP is required.
b. After multiplying by the total number of samples (15 samples + 1 negative
control), 320 μL of 10.0 mM PNPP and 2720 μL of Tris are required.
c. Also these values are multiplied by 1.2, which requires 384 μL of 10.0 mM PNPP
and 3264 μL of Tris.
5. You will test each sample in duplicate in order to produce more reliable and accurate
results. In order to account for this, the above volumes of PNPP and Tris have been
multiplied by2.
6. Mix768 μL 10.00 mM PNPP and 6528 μL 10.0 mM TrispH 9together (total 7296
μL) in a 15mL tube. You should observe a colorless or pale yellow solution.
7. Pipette 190 µL of the PNPP/Tris mixture into wells of a 96 well plate in sets of 2 (e.g.
A1, A2)
8. Determine the couple of wells that you wish to use as your negative control. Add 10 µL
of 5.0 mM Tris pH 7.4 buffer to each of these wells. Make a grid on benchling to keep
track of which well has which solution. A sample benchling grid is provided below:
9 . Pipette 200 µL of your positive control into two empty wells
10.Quickly add 10 µL of each sample into two sets of wells. The reaction will begin
immediately so load the plate into the BioTek reader as soon as possible.
11.Read the absorbance levels of the plate at 400 nm. The program will read the absorbance
of each sample at zero and five minutes. Copy down the five minute absorbance reading
for the calculation in your DLN.
12.Check to see if the absorbance of your sample wells has increased from zero to five
minutes.
a. If some of your samples don’t show a change, then this indicates no enzyme
activity in that respective well or you waited too long between adding your
samples and reading the plate.
13.Average the duplicate absorbances for each sample and control at five minutes. Subtract
the average absorbance of the negative control from the average absorbance from each
sample including the positive control.
a. Negative values after this subtraction indicate no enzyme activity. If all are
negative, then an error has occurred during the experiment. Re-do the protocol.
Part 2 - Invertase Activity Assay
1. Dilute 0.100 M sucrose to 0.020 M in a final volume of 1.0mL using200 μl of 0.100 M
sucrose and 800 μl of 150.0 mM acetate pH 4.8
2. Prepare both positive and negative controls
a. Positive: using 10.0µL 5.0 mM Tris-Cl pH 7.4 and100 µL 0.020M glucose +
fructose in a microcentrifuge tube. Label the tube as the positive control
b. Negative: In a microcentrifuge tube, combine 10.0 µL of 5 mM Tris-Cl pH 7.4
and 100 µL 0.020 M prepared dilute sucrose solution. Label the microcentrifuge
tube accordingly.
3. For all 15 samples, use the following procedure:
a. In a microcentrifuge tube, combine 10.0 µL of the sample liquid with 100 µL of
the dilute 0.020 M sucrose solution in intervals of every five seconds. Label the
microcentrifuge tube accordingly.
b. **Begin recording the time using a stopwatch from the addition of the sucrose
now to the addition of the alkaline DNS later on in the procedure. Notate this in
your lab notebook as the reaction time. **
c. Before adding DNS, allow all 15 samples, now combined with dilute sucrose, to
rest on the bench for around five minutes.
d. Add alkaline DNS to each tube, in intervals of every five seconds to keep
additions consistent.
e. Heat all tubes at 90 degrees celsius for 5 minutes.
f . A dd 400 µL 50.0 mM acetate pH 4.8 to each tube.
g. Pipet 200 µL of each sample into a 96 well plate in duplicates. Be sure to notate
in your notebook exactly how the layout was set up. An image of one possible
layout has been provided below:
. P
h ut the plate into the absorbance machine and read it at 540 nm.
i. Ensure that the absorbance of the positive control is different from the negative
control. Re-do the process if you suspect something is wrong with your samples.
j. Average the absorbance values provided for each sample, subtract the average
absorbance of the negative control to check that your samples are above the
negative control’s level of enzyme activity. Re-do the process if this is not the
case.
Part 3 - Protein Quantification
1. Quantify the total amount of proteins in each of your 15 samples on the nanodrop.
2. Pipet each 2 µL of the 15 samples on the instrument and measure the data in mg/mL.
3. Record the quantification data of the 15 samples in your lab notebook.
