ab139462
CD45 Tyrosine
Phosphatase Activity
Assay Kit (Colorimetric)
Instructions for Use
For the screening of inhibitors and modulators of
CD45 phosphatase activity
This product is for research use only and is not
intended for diagnostic use.
Version 3 Last Updated 8 April 2015
Table of Contents
1.
Principle of the Assay
2
2.
Protocol Summary
3
3.
Materials Supplied
5
4.
Storage and Stability
6
5.
Materials Required, Not Supplied
8
6.
Assay Protocol
9
7.
Data Analysis
15
1
1. Principle of the Assay
Abcam CD45 Tyrosine Phosphatase Activity Assay Kit (Colorimetric)
(ab139462) is a colorimetric, non-radioactive assay designed for a
microplate format. It may be used to screen inhibitors and
modulators of CD45 phosphatase activity, and/or to study other
aspects of CD45 enzyme kinetics.
The kit includes purified Human CD45 cytoplasmic domain (residues
584-1281; MW=95 kDa), expressed in a yeast expression system.
The basis for the assay is the cleavage of a synthetic
phosphotyrosine peptide based on the sequence of the negative
regulatory site of pp60c-src. The phosphate released due to
enzymatic cleavage is quantified using the Green Assay Reagent in
a modified Malachite Green assay.
A CD45 tyrosine phosphatase inhibitor, RWJ-60475, is included as a
test control for inhibitor screening. It has a reported IC50 = 2 µM.
RWJ-60475 is a hydrolytically stable bioisostere of phosphotyrosine
that binds to the catalytic site of CD45. It is not cell permeable. The
assay offers the following advantages:
1) Non-radioactive
2) Convenient one step detection
3) Microplate format
2
2. Protocol Summary
Prepare Standard curve
Dilute standard
Prepare standard wells
Add Green Assay Reagent
Time course/linearity assay
Dilute CD45 Tyrosine Phosphatase substrate
Add CD45 Tyrosine Phosphatase substrate to appropriate wells
Add assay buffer to each well
Add CD45 enzyme to each well at time intervals
Terminate reaction by adding Green Assay Reagent
3
Test sample/inhibitor assay
Prepare samples containing CD45 enzyme, substrate and test
compound. Include RWJ-60475 (optional)
Incubate samples at appropriate temperature and time
Terminate reaction by adding Green Assay Reagent
4
3. Materials Supplied
Item
Quantity
Storage
CD45 Enzyme (Human, Recombinant)
1 x 50 µL
-80°C
1 x 1 mg
-80°C
1 vial
-80°C
1 x 20 mL
-80°C
Green Assay Reagent
1 x 20 mL
4°C
Phosphate Standard (80µM)
1 x 0.5 mL
-80°C
1
4°C
(300U/µL; MW=95kDa)
RWJ-60475 Inhibitor (MW:404.1)
CD45 Tyrosine Phosphatase Substrate
(MW:1543.7; lyophilized)
CD45 Tyrosine Phosphatase 2X Assay Buffer
(100mM HEPES, pH7.2, 2mM EDTA, 2mM
DTT, 0.1%NP-40)
96-well Clear Microplate (½ Volume)
5
4. Storage and Stability

Store all components, except the microtiter plate and Green
Assay Reagent, at -80°C for the highest stability. The CD45
enzyme component must be handled particularly carefully in
order to retain maximal enzymatic activity. Thaw it quickly in a
RT water bath or by rubbing between fingers, then immediately
store on an ice bath. The remaining unused enzyme should be
quickly refrozen, for example in liquid N2 or dry ice/ethanol, and
stored at -80°C.

Because its stability when frozen in solution is poor, the RWJ60475 Inhibitor is provided as lyophilized aliquot. For highest
activity, we recommend to dissolve in DMSO or absolute ethanol
(max. solubility=25mg/mL)

One U of CD45 enzyme =1pmol per minute under the conditions
of the linearity assay. One unit will hydrolyze 1 nmol pnitrophenyl phosphate (pNPP) per minute in 1X Assay Buffer
with 50mM pNPP, at pH 7.2 and 30°C.
6

The Green Assay Reagent is a highly sensitive phosphate
detection solution. Free phosphate present on labware and in
reagent
solutions
will
greatly
increase
the
background
absorbance of the assay. This is detected visually as a change
in color from yellow to green. Detergents used to clean labware
may contain high levels of phosphate. Use caution by either
rinsing labware with diH2O or employ unused plasticware.
7
5. Materials Required, Not Supplied

Microplate reader capable of reading OD620nm

Pipettes or multi-channel pipettes capable of pipetting 5-100 µL
accurately.
(Note: reagents can be diluted to increase the minimal pipetting
volume to >10 µL).

Ice bucket to keep reagents cold until use.

Water bath or incubator for component temperature equilibration.

DMSO or absolute ethanol

Orbital shaker
8
6. Assay Protocol
A. Reagent Preparation
1. Thaw assay buffer, CD45 enzyme, the CD45 Tyrosine
Phosphatase Substrate, RWJ-60475 inhibitor, Phosphate
standard. Store all on ice.
2. Reconstitute CD45 Tyrosine Phosphatase substrate to 1 mM
by adding 324 µL of 2X assay buffer plus 324 µL dH2O.
Vortex. After use, store remaining substrate at -80°C.
3. Optional: Dissolve RWJ-60475 inhibitor in DMSO or absolute
ethanol to desired molarity (MW:404.1g/mol)
4. Warm Green Assay Reagent to room temperature.
B. Preparing a Standard Curve
1. Prepare 1 mL 1X assay buffer for standard curve dilutions
(dilute 500 µL of 2X assay buffer with 500µL of dH2O).
2. Dilute phosphate standard to 50 µM with assay buffer. For
example, add 125 µL phosphate standard (80 µM) and bring
up to 200 µL with 1X assay buffer.
3. Prepare 1:1 serial dilutions of phosphate standard, using
assay buffer, plus an assay buffer blank (40 µL per well).
Concentrations of 50, 25, 12.5, 6.25, 3.125, 1.5625 and
0.7813 µM correspond to 2, 1, 0.5, 0.25, 0.125, 0.063 and
0.031 nmol/well PO4 (see Table 1):
9
a) Add 80 µL of 50 μM phosphate standard (Step 1) to well
A of assay plate and 80 µL of 50 μM standard to well B.
b) Add 40 µL 1X assay buffer to wells C through H.
c) Remove 40 µL from well A and add it to well C. Mix
thoroughly by pipetting up and down several times.
d) Remove 40 µL from well C and add it to well E. Mix well
E thoroughly and then remove 40 µL and discard.
e) Remove 40 µL from well B and add it to well D. Mix
thoroughly by pipetting up and down several times.
Repeat this process moving and mixing 40 µL from well
D to F and then from F to G. Remove and discard 40 µL
from well G. DO NOT PROCEED TO THE BLANK
WELL ‘H’.
10
Table 1. Layout example of time course/linearity assay.
Phosphate Standard
Sample Well
Curve nmol
(Columns 1,2)
Time course
Min. (Columns 3,4)
A
2
60
B
1
45
C
0.5
30
D
0.25
20
E
0.125
10
F
0.063
5
G
0.031
2
H
0
0
For highest accuracy, perform all samples in duplicate.
See Figures 1 and 2 for example results.
C. Preparing a Time Course/Linearity Assay
1. Add 8 µL of CD45 Tyrosine Phosphatase substrate (1 mM;
Step
6.A.2)
to
appropriate
wells
(
final
substrate
concentration in the well should be 200 µM).
2. Add 27 µL 1X assay buffer to each well.
3. Designate a reaction time to each well (e.g.: 60, 45, 30, 20,
10, 5, 2 and 0 min). See Table 1.
11
4. Equilibrate microplate to desired reaction temperature (e.g.:
30°C).
5. Prepare CD45 enzyme 1/20 into 1X assay buffer (e.g.: 5 µL
CD45 + 95 µL assay buffer), making enough for the assays
planned. Each well will receive 5 µL. Store dilution on ice.
6. Start reactions by addition of 5 µL of CD45 enzyme. Total
CD45 enzyme= 75 U/well. Make the additions in the reverse
time order such that all incubations end at the same time
(e.g.: Add 60 min time pt. at t=0; add 5 min at t=55 min, etc.).
The total reaction volume=40 µL.
12
D. Preparing a Test Sample/Inhibitor Assay
1. Prepare samples containing CD45 enzyme, substrate and
test compound dissolved in 1x Assay Buffer as listed in
Table 2. Include the RWJ-60475 Inhibitor if desired.
2. Incubate samples at desired temperature (e.g. 37°C) and
time (e.g.: 60 min).
Table 2: Example of Test Samples/Inhibitor Assay Microplate Samples
CD45
Substrate
Assay
Test
(1 mM)
Buffer
compound
Control
8 µL
27 µL
0 µL
5 µL
Test
8 µL
0 µL
27 µL
5 µL
8 µL
0 µL
27 µL
5 µL
RWJ-60475
Inhibitor
Enzyme
(15U/ µL)
3. To confirm that an apparent RWJ-60475 inhibitor does not
interfere with the release of phosphate by CD45, test
additional wells using phosphate standard and the inhibitor
in question. Compare the results with the 50 μM standard
curve well.
4. Add additional controls as necessary. For example, the test
compound may interfere with Green Assay Reagent
development. In this case, include a sample containing the
test compound with no CD45 (add CD45 buffer in its place).
13
NOTE:
1. For highest accuracy, perform all samples in duplicate.
2. Final assay conditions are 75U/well CD45, 200 µM
substrate in total assay volume of 40 µL.
3. See Figures 3 and 4 for example results.
E. Terminating Reactions
1. After incubating wells for desired duration, terminate
reactions and visualize phosphate by addition of 100 µL
Green Assay Reagent. Agitate plate or triturate wells gently
to mix.
Note: Avoid production of air bubbles in the wells.
2. Allow color to develop for 20-30 minutes. Be careful to
assure samples spend approximately the same time with the
reagent before reading on the microplate reader.
3. Read OD620nm on microtiter-plate reader.
Note: Retain microtiter plate for future use of unused wells.
14
7. Data Analysis
A. Phosphate Standard Curve
1. Plot standard curve data as OD620nm versus nmol Phosphate
(see Fig. 1).
2. Fit a line to the plotted data using an appropriate linear
regression program.
3. Rearrange the equation for best-fit line to solve for nmol of
Phosphate in terms of OD620nm.
Phosphate released = (OD620nm – y-intercept)/slope
4. Substitute OD620nm data obtained from experimental samples
(e.g. a CD45 reaction) into the rearranged equation to obtain the
µM (or nmol/well) of phosphate produced.
0.6
O.D.(620nm)
0.5
0.4
0.3
0.2
0.1
0.0
0
20
40
60
-3
PO4 (µM)
Figure 1. Example data for a standard curve for phosphate. Duplicate
wells of phosphate dilutions as described in Section 6B.
15
B. Determining the linear range of a CD45 reaction time course
Determine the reaction time range in which the amount of phosphate
released is linear. In Figure 2, this range is from 0-60 minutes. This
value
is
variable
depending
on
reaction
conditions
and
storage/handling of the CD45. The time range can be lengthened by
decreasing the amount of CD45 in the assay and lowering the assay
temperature. For accurate results, it is important to perform
inhibitor/agonist assays under linear assay conditions.
Time course
(P301 substrate 200µM,
CD45 15U/µL)
O.D.(620nm)
0.8
0.6
0.4
0.2
0.0
0
20
40
60
80
Time (min.)
Figure 2. Example of an average time course plot of CD45. Duplicate
wells of time course/linearity assay as described in Section 6C. (performed
at room temperature).
16
C.Determine activity% of test sample/Inhibitor
If the 0 time (Table 1, well# H3,4) has a significant value, subtract
this number from all samples readings.
Calculate % activity:
%activity = (OD of test sample/OD of control) x 100
% Enzyme Activity
150
100
50
R
W
(1 J-6
2. 04
5µ 7
M 5
)
N
a
(1 3V
5µ O4
M
)
C
on
tr
ol
0
Figure 3. Example of calculated inhibition. Raw data obtained from test
sample/inhibitor assay as described in Section 6D. (performed at room
temperature)
17
% Enzyme Activity
100
80
60
40
20
0
0.1
1
10
100
RWJ-60475 (µM)
1000
Figure 4. Example of calculated RWJ-60475 inhibition curve (detailed)
Raw data obtained from test sample/inhibitor assay as described in Section
6D. (performed at room temperature)
% Enzyme Activity
15
10
5
0
1
10
100
1000
Na-orthovanadate (µM)
Figure 5. Example of calculated Na3VO4 inhibition curve (detailed)
Raw data obtained from test sample/inhibitor assay as described in Section
6D. (performed at room temperature)
18
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19
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