ab168541 –
Active Caspase 3
(Asp175) Human ELISA
Kit
Instructions for Use
For the quantitative measurement of p17 subunit of active caspase 3 in
human cells and tissue homogenates.
This product is for research use only and is not intended for diagnostic
use.
Version 1 Last Updated 12 December 2014
Table of Contents
INTRODUCTION
1.
BACKGROUND
2.
ASSAY SUMMARY
2
4
GENERAL INFORMATION
3.
PRECAUTIONS
4.
STORAGE AND STABILITY
5.
MATERIALS SUPPLIED
6.
MATERIALS REQUIRED, NOT SUPPLIED
7.
LIMITATIONS
8.
TECHNICAL HINTS
5
5
5
6
6
7
ASSAY PREPARATION
9.
REAGENT PREPARATION
10. STANDARD PREPARATION
11. SAMPLE PREPARATION
12. PLATE PREPARATION
8
8
10
11
ASSAY PROCEDURE
13. ASSAY PROCEDURE
12
DATA ANALYSIS
14. CALCULATIONS
15. TYPICAL DATA
16. TYPICAL SAMPLE VALUES
17. SPECIES REACTIVITY
18. ASSAY SPECIFICITY
14
15
17
18
18
RESOURCES
19.
20.
TROUBLESHOOTING
NOTES
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20
21
1
INTRODUCTION
1.
BACKGROUND
Abcam’s Active Caspase 3 (Asp175) Human in vitro ELISA (EnzymeLinked Immunosorbent Assay) kit is designed for the accurate
quantitative measurement of p17 subunit of active caspase 3 protein in
human cells and tissue homogenates.
Caspase 3 is a cysteine protease involved in the activation cascade of
caspases responsible for apoptosis execution. At the onset of
apoptosis caspase 3 proteolytically cleaves poly (ADP-ribose)
polymerase (PARP) at Asp216-Gly217 bond. Caspase 3 cleaves and
activates sterol regulatory element binding proteins (SREBPs) between
the basic helix-loop-helix leucine zipper domain and the membrane
attachment domain. Caspase 3 cleaves and activates caspase-6, -7
and -9. Caspase 3 is involved in the cleavage of huntingtin. Caspase 3
is a cytoplasmic protein highly expressed in lung, spleen, heart, liver
and kidney. Moderate levels of caspase 3 are in brain and skeletal
muscle, and low levels in testis. Also caspase 3 is found in many cell
lines, highest expression in cells of the immune system.
Caspase 3 is expressed in an inactive pro-form (pro caspase 3). In
apoptosis, the pro caspase 3 is activated by proteolytic cleavages at
Asp28-Ser29 and Asp175-Ser176 bonds catalyzed by granzyme B,
caspase-6, caspase-8, caspase-9 and caspase-10 generating two
active subunits. Thus the pro-form and the active form are useful
biomarkers of apoptosis. Active caspase 3 is a heterotetramer that
consists of two anti-parallel arranged heterodimers, each one formed
by a 17 kDa (p17) and a 12 kDa (p12) subunit. Additional processing of
the propeptides is likely due to the autocatalytic activity of the activated
protease. Active heterodimers between the small subunit of caspase-7
protease and the large subunit of caspase-3 also occur and vice versa.
Caspase 3 is S-nitrosylated on its catalytic site cysteine in
unstimulated human cell lines and denitrosylated upon activation of the
Fas apoptotic pathway, associated with an increase in intracellular
caspase activity. Fas therefore activates caspase-3 not only by
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2
INTRODUCTION
inducing the cleavage of the caspase zymogen to its active subunits,
but also by stimulating the denitrosylation of its active site thiol.
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3
INTRODUCTION
2. ASSAY SUMMARY
Remove appropriate number of
antibody coated well strips. Equilibrate
all reagents to room temperature.
Prepare all the reagents, samples, and
standards as instructed.
Add standard or sample to each well
used. Incubate at room temperature.
Aspirate and wash each well. Add
prepared Detector Antibody to each
well. Incubate at room temperature.
Aspirate and wash each well. Add
prepared HRP label. Incubate at room
temperature.
Aspirate and wash each well. Add
TMB Development Solution to each
well. Immediately begin recording the
color development. Alternatively add a
Stop solution at a user-defined time.
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4
GENERAL INFORMATION
3. PRECAUTIONS
Please read these instructions carefully prior to beginning the
assay.
All kit components have been formulated and quality control tested to
function successfully as a kit. Modifications to the kit components or
procedures may result in loss of performance.
4. STORAGE AND STABILITY
Store kit at 4ºC immediately upon receipt.
Refer to list of materials supplied for storage conditions of individual
components. Observe the storage conditions for individual prepared
components in section 9 & 10.
5. MATERIALS SUPPLIED
Item
2X Extraction Buffer
20X Buffer
10X Blocking Solution
10X Wash Buffer
Caspase 3 Microplate
HeLa-Staurosporine Standard
10X Caspase 3 (Asp175) Detector Antibody
10X HRP Label
TMB Development Solution
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Amount
Storage
Condition
(Before
Preparation)
15 mL
3 mL
4ºC
4ºC
6 mL
40 mL
96 Wells
400 µg
4ºC
4ºC
4ºC
4ºC
4ºC
4ºC
4ºC
700 µL
1 mL
12 mL
5
GENERAL INFORMATION
6. MATERIALS REQUIRED, NOT SUPPLIED
These materials are not included in the kit, but will be required to
successfully utilize this assay:

Microplate reader capable of measuring absorbance at 600 or
450 nm.

Method for determining protein concentration (BCA assay
recommended).

Deionized water.

Multi and single channel pipettes

PBS (1.4 mM KH2PO4, 8 mM Na2HPO4, 140 mM NaCl, 2.7 mM
KCl, pH 7.3).

Tubes for standard dilution.

Stop solution (optional) – 1N Hydrochloric acid (HCl).

Plate shaker for all incubation steps (optional).

Phenylmethylsulfonyl
inhibitors).
Fluoride
(PMSF)
(or
other
protease
7. LIMITATIONS

Assay kit intended for research use only. Not for use in diagnostic
procedures.

Do not use kit or components if it has exceeded the expiration date
on the kit labels.

Do not mix or substitute reagents or materials from other kit lots or
vendors. Kits are QC tested as a set of components and
performance cannot be guaranteed if utilized separately or
substituted.
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6
GENERAL INFORMATION
8. TECHNICAL HINTS

Samples generating values higher than the highest standard
should be further diluted in the appropriate sample dilution buffers

Avoid foaming
components.

Avoid cross contamination of samples or reagents by changing tips
between sample, standard and reagent additions.

Ensure plates are properly sealed or covered during incubation
steps.

Complete removal of all solutions and buffers during wash steps is
necessary to minimize background.

As a guide, typical ranges of sample concentration for commonly
used sample types are shown below in Sample Preparation
(section 11).

All samples should be mixed thoroughly and gently.

Avoid multiply freeze/thaw of samples.

Incubate ELISA plates on a plate shaker during all incubation
steps (optional).

When generating positive control samples, it is advisable to
change pipette tips after each step.
or
bubbles
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when
mixing
or
reconstituting
7
ASSAY PREPARATION
9. REAGENT PREPARATION
Equilibrate all reagents to room temperature (18-25°C) prior to use.
9.1
1X Extraction Buffer
If preparing extracts from cell pellets (11.1) prepare
1X Extraction Buffer by adding 15 mL 2X Extraction Buffer to
15 mL nanopure water plus protease inhibitors.
9.2
Incubation Buffer
Prepare Incubation Buffer by adding 1.5 mL 20X Buffer and
3 mL 10X Blocking Solution to 25.5 mL nanopure water. Mix
gently and thoroughly.
9.3
1X Wash Buffer
Prepare 1X Wash Buffer by adding 40 mL 10X Wash Buffer
to 360 mL nanopure water. Mix gently and thoroughly.
9.4
1X Active Caspase 3 (Asp175) Detector Antibody
Prepare 1X Active Caspase 3 (Asp175) Detector Antibody
by diluting the 10X Active Caspase 3 (Asp175) Detector
Antibody 10-fold with Incubation Buffer immediately prior to
use. Prepare 500 µL 1X Active Caspase 3 (Asp175)
Detector Antibody for each 8 well strip used.
9.5
1X HRP Label
Prepare 1X HRP Label by diluting the 10X HRP Label 10fold with Incubation Buffer immediately prior to use. Prepare
500 µL 1X HRP Label for each 8 well strip used.
● After opening, the unused Incubation Buffer should be stored at
-20°C.
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8
ASSAY PREPARATION
10. STANDARD PREPARATION
Prepare serially diluted standards immediately prior to use. Always
prepare a fresh set of positive controls for every use.
10.1 Reconstitute the standard sample by adding 400 µL
1X Incubation Buffer to HeLa-Staurosporine Standard. Allow
to sit for 5 minutes on ice. Mix gently and thoroughly. This
1,000 µg/mL stock = Standard #1 (see table below). Any
remaining stock material should be stored at -80°C.
10.2 Label tubes #2-8: Add 150 μL Incubation Buffer into each
tube.
10.3 Add 150 μL Standard #1 to tube #2 = Standard #2. Mix
gently and thoroughly.
10.4 Transfer 150 μL from Standard #2 to tube #3, = Standard
#3. Mix gently and thoroughly.
10.5 Using the table below as a guide, repeat for Tubes #4
through #7.
10.6 Use the diluent as the zero standard tube labeled #8.
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9
ASSAY PREPARATION
Standard
#
Sample to
Dilute
Volume to
Dilute
(µL)
1
2
3
4
5
6
7
Stock
Standard #1
Standard #2
Standard #3
Standard #4
Standard #5
Standard #6
300
150
150
150
150
150
150
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Volume
of
Diluent
(µL)
0
150
150
150
150
150
150
Starting
Conc.
(µg/mL)
Final
Conc.
(g/mL)
1,000
1,000
500
250
125
62.5
31.25
1,000
500
250
125
62.5
31.25
15.63
10
ASSAY PREPARATION
11. SAMPLE PREPARATION
TYPICAL SAMPLE DYNAMIC RANGE Typical working ranges
Sample Type
Range
HeLa cells treated for 4 hours
with 1 µM staurosporine
16-1,000 µg/mL
Jurkat cells treated for 4 hours
with 1 µM staurosporine
4-250 µg/mL
11.1 Preparation of extracts from cell pellets
11.1.1 Collect non adherent cells by centrifugation or
scrape to collect adherent cells from the culture
flask. Typical centrifugation conditions for cells are
500 x g for 5 minutes at 4ºC.
11.1.2 Rinse cells twice with PBS.
11.1.3 Solubilize cell pellet at 2x107/mL in 1X Extraction
Buffer
11.1.4 Incubate on ice for 20 minutes. Centrifuge at
18,000 x g for 20 minutes at 4°C. Transfer the
supernatants into clean tubes and discard the
pellets. Assay samples immediately or aliquot and
store at -80°C. The sample protein concentration in
the extract may be quantified using a protein assay.
11.2 Preparation of lysates from cells in media (in-well lysis)
11.2.1 Seed cells at the same density into a multi-well plate
(e.g. 96-well plate) and treat them as desired.
11.2.2 Solubilize the cells by adding equal volume (equal to
the volume of culture media) of 2X Extraction Buffer
(supplemented with 2X protease inhibitors) directly
to the cells in growth media.
11.2.3 Incubate on ice for 20 minutes. If available use a
plate shaker at 300 rpm.
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11
ASSAY PREPARATION
11.2.4 Assay samples immediately or aliquot and store at
-80°.
11.3 Preparation of extracts from tissue homogenates
11.3.1 Tissue lysates are typically prepared by
homogenization of tissue that is first minced and
thoroughly rinsed in PBS to remove blood (dounce
homogenizer recommended).
11.3.2 Suspend the homogenate to 10 mg/mL in PBS.
11.3.3 Solubilize the homogenate by combining equal
volumes of 2X Extraction Buffer and the
homogenate.
11.3.4 Incubate on ice for 20 minutes. Centrifuge at
18,000 x g for 20 minutes at 4°C. Transfer the
supernatants into clean tubes and discard the
pellets. Assay samples immediately or aliquot and
store at -80°C. The sample protein concentration in
the extract may be quantified using a protein assay.
●
The samples should be diluted to within the working range of the
assay in Incubation Buffer.
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12
ASSAY PREPARATION
12. PLATE PREPARATION
●
●
The 96 well plate strips included with this kit are supplied ready to
use. It is not necessary to rinse the plate prior to adding reagents.
Unused plate strips should be returned to the plate packet and
stored at 4°C.
●
For each assay performed, a minimum of 2 wells must be used as
the zero control.
●
For statistical reasons, we recommend each sample should be
assayed with a minimum of two replicates (duplicates).
●
Well effects have not been observed with this assay. Contents of
each well can be recorded on the template sheet included in the
Resources section.
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13
ASSAY PROCEDURE
13. ASSAY PROCEDURE
●
Equilibrate all materials and prepared reagents to room
temperature prior to use.
●
It is recommended to assay all standards, controls and
samples in duplicate.
13.1 Prepare all reagents, working standards, and samples as
directed in the previous sections.
13.2 Remove excess microplate strips from the plate frame,
return them to the foil pouch containing the desiccant pack,
and reseal.
13.3 Add 50 µL of each sample per well. It is recommended to
include a dilution series of standard protein (section 10), as
well as untreated sample. Also include a no material control
as a zero standard.
13.4 Cover/seal the plate and incubate for 2 hours at room
temperature.
If available use a plate shaker for all
incubation steps at 300 rpm.
13.5 Aspirate each well and wash, repeat this once more for a
total of two washes. Wash by aspirating or decanting from
wells then dispensing 300 µL 1X Wash Buffer into each well
as described above. Complete removal of liquid at each
step is essential to good performance. After the last wash,
remove the remaining buffer by aspiration or decanting.
Invert the plate and blot it against clean paper towels to
remove excess liquid.
13.6 Immediately before use prepare sufficient (500 µL/8 well
strip used) 1X Active Caspase 3 (Asp175) Detector Antibody
in Incubation Buffer (step 9.4). Add 50 µL 1X Detector
antibody to each well used. Cover/seal the plate and
incubate for 1 hour at room temperature. If available use a
plate shaker for all incubation steps at 300 rpm.
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14
ASSAY PROCEDURE
13.7 Repeat the aspirate/wash procedure above.
13.8 Immediately before use prepare sufficient (500 µL/strip
used) 1X HRP Label in Incubation Buffer (step 9.5). Add
50 µL 1X HRP Label to each well used. Cover/seal the plate
and incubate for 1 hour at room temperature. If available
use a plate shaker for all incubation steps at 300 rpm.
13.9 Repeat the aspirate/wash procedure above, however,
performing a total of three washes.
13.10 Add 100 µL TMB Development Solution to each empty well
and immediately begin recording the blue color development
with elapsed time in the microplate reader prepared with the
following settings:
Mode:
Kinetic
Wavelength:
600 nm
Time:
up to 15 min
Interval:
20 sec - 1 min
Shaking:
Shake between readings
Alternative– In place of a kinetic reading, at a user defined,
time record the endpoint OD data at (i) 600 nm or (ii) stop
the reaction by adding 100 µL Stop solution (1N HCl) to
each well and record the OD at 450 nm.
13.11 Analyze the data as described below.
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15
DATA ANALYSIS
14. CALCULATIONS
Subtract average zero standard reading from all readings. Average the
duplicate readings of the positive control dilutions and plot against their
concentrations. Draw the best smooth curve through these points to
construct a standard curve. Most plate reader software or graphing
software can plot these values and curve fit. A four parameter
algorithm (4PL) usually provides the best fit, though other equations
can be examined to see which provides the most accurate (e.g. linear,
semi-log, log/log, 4 parameter logistic).
Read relative protein
concentrations for unknown samples from the standard curve plotted.
Samples producing signals greater than that of the highest standard
should be further diluted and reanalyzed, then multiplying the
concentration found by the appropriate dilution factor.
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16
DATA ANALYSIS
15. TYPICAL DATA
TYPICAL STANDARD CURVE – Data provided for demonstration
purposes only. A new standard curve must be generated for each
assay performed.
Standard Curve Measurements
Conc.
(µg/mL)
Change mOD/min (600 nm)
1
2
3
Mean
0.00
0.34
0.58
0.46
0.46
15.63
2.12
2.13
1.96
2.07
31.25
3.80
3.67
3.43
3.63
62.50
7.23
7.00
6.81
7.01
125.00
13.57
13.31
13.05
13.31
250.00
25.04
26.22
25.68
25.65
500.00
46.87
49.96
48.07
48.30
1,000.00
80.03
80.87
81.53
80.81
Figure 1. Example HeLa-Staurosporine standard curve. The
HeLa-Staurosporine standard was prepared using HeLa cells treated for 4
hours with 1 µM staurosporine (ab120056). Raw data values are shown in the
table. Background-subtracted data values (mean +/- SD) are graphed.
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17
DATA ANALYSIS
Figure 2. Example of staurosporine IC50 determination. Lysates
corresponding to 0.33x106 HeLa cells/mL or 0.50x106 Jurkat cells/mL were
prepared by direct in-well lysis (without media removal) from cells treated for 4
hours with variable doses of staurosporine in a 96-well plate. 100 µL of the
lysates were analyzed in triplicates. Background-subtracted signals are shown
in left panels. Relative active caspase 3 concentrations interpolated from
standard curves and expressed as percent of cells treated with the highest
dose of staurosporine are shown in right panels. IC50 were 0.9 µM for HeLa
cells and 0.4 µM for Jurkat cells.
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18
DATA ANALYSIS
16. TYPICAL SAMPLE VALUES
SENSITIVITY Calculated minimum detectable dose = 2 µg/mL (zero dose n = 25 + 2
standard deviations) using HeLa-Staurosporine Standard.
RECOVERY –
(Sample spiking in representative sample matrices)
Sample Type
50% culture media
(10FHGDMEM)
10% goat serum
50% Extraction Buffer
Average %
Recovery
Range
104
98 -109
93
92 -93
107
106 -109
LINEARITY OF DILUTION –
Linearity of dilution was determined by comparing dilution series of
extracts prepared from staurosporine-treated Jurkat cells to dilution
series of the HeLa-Staurosporine Standard.
JurkatStaurosporine
Dilution
(fold)
JurkatStaurosporine
(µg/mL)
Undiluted
2
4
8
16
32
64
250.00
125.00
62.50
31.25
15.63
7.81
3.91
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Interpolated
Relative Value
1,053
490
240
115
64
32
16
% Expected
Value
100
93
91
88
98
97
95
19
DATA ANALYSIS
PRECISION –
Mean of coefficient of variations within working range using
HeLa-Staurosporine Standard.
n=
%CV
Intra-Assay
3
3.4
Inter-Assay
3
6.7
17. SPECIES REACTIVITY
This kit detects p17 subunit of active caspase 3 in human samples
only.
18. ASSAY SPECIFICITY
Western blot analysis of the proteins immunocaptured with the use of
the Caspase 3 Microplate shows that the microplate captures both the
pro- and the p17 subunit of caspase 3 (Figure 3). Using the assay kit,
strong signals were detected in Jurkat and HeLa cells treated with
staurosporine, a drug known to induce apoptosis involving cleavage of
pro caspase 3 into p17 and p12 subunits (Figure 4). No detectable
signal was found in vehicle treated cells (Figure 4). Taken together,
these results demonstrate that this assay kit specifically measures the
p17 subunit of the active caspase 3 protein.
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20
DATA ANALYSIS
1
2
3
4
250 -150 -100 -75 -50 -37 -25 -20 -15 -10 --
Figure 3. Demonstration of the capture antibody specificity. Cell extracts
were prepared from 4 hours vehicle-treated (lanes 1 and 3) and 1 µM
staurosporine-treated (lanes 2 and 4) Jurkat cells. Extracts were incubated
with the Caspase 3 Microplate, captured proteins were extracted and analyzed
by Western blotting using a pro+p17 caspase 3 antibody ab32351 (Lanes 3
and 4). 25% of the amounts of the extracts used for immunocapture were also
analyzed directly by the Western blotting (lanes 1 and 2). Note that the
Caspase 3 Microplate captures both the pro- and p17 subunit of caspase 3.
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21
DATA ANALYSIS
Active Caspase 3
(Relative)
500
400
Vehicle
Staurosporine
300
200
100
0
HeLa
Jurkat
Figure 4. Demonstration of assay specificity. Demonstration of the assay
specificity by induction of active caspase 3 by staurosporine treatment. Cells
were treated for 4 hours with 1 µM staurosporine or drug’s vehicle (DMSO)
and 500 µg/mL of HeLa cell extracts and 125 g/mL Jurkat cell extracts
prepared from cell pellets were analyzed using this kit. Relative active caspase
3 levels were interpolated from HeLa-Staurosporine standard curve and
expressed as percent of staurosporine-treated HeLa cell. Note that no active
caspase 3 (p17 subunit) was detected in vehicle-treated HeLa and Jurkat cells.
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22
RESOURCES
19. TROUBLESHOOTING
Problem
Poor standard
curve
Low Signal
Large CV
Low sensitivity
Cause
Solution
Inaccurate Pipetting
Check pipets
Improper standard
dilution
Prior to opening, briefly spin
the stock standard tube and
dissolve the powder
thoroughly by gentle mixing
Incubation times too
brief
Ensure sufficient incubation
times; change to overnight
standard/sample incubation
Inadequate reagent
volumes or improper
dilution
Check pipettes and ensure
correct preparation
Incubation times with
TMB too brief
Ensure sufficient incubation
time till blue color develops
prior addition of Stop solution
Plate is insufficiently
washed
Review manual for proper
wash technique. If using a
plate washer, check all ports
for obstructions
Contaminated wash
buffer
Make fresh wash buffer
Improper storage of
the ELISA kit
Store your reconstituted
standards at -80°C, all other
assay components 4°C.
Keep substrate solution
protected from light
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RESOURCES
20. NOTES
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RESOURCES
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RESOURCES
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26
UK, EU and ROW
Email: technical@abcam.com | Tel: +44-(0)1223-696000
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Email: wissenschaftlicherdienst@abcam.com | Tel: 019-288-259
France
Email: supportscientifique@abcam.com | Tel: 01-46-94-62-96
Germany
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Spain
Email: soportecientifico@abcam.com | Tel: 911-146-554
Switzerland
Email: technical@abcam.com
Tel (Deutsch): 0435-016-424 | Tel (Français): 0615-000-530
US and Latin America
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Canada
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China and Asia Pacific
Email: hk.technical@abcam.com | Tel: 108008523689 (中國聯通)
Japan
Email: technical@abcam.co.jp | Tel: +81-(0)3-6231-0940
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All information / detail is correct at time of going to print.
RESOURCES
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