ab128499 – Estrogen
Receptor Alpha ELISA
Kit
Instructions for Use
For the quantitative measurement of Human, mouse and hamster
Estrogen Receptor Alpha concentrations in cell and nuclear extracts.
This product is for research use only and is not intended for diagnostic
use.
Version 3 Last Updated 2 December 2014
Table of Contents
INTRODUCTION
1.
BACKGROUND
2
2.
ASSAY SUMMARY
4
GENERAL INFORMATION
3.
PRECAUTIONS
5
4.
STORAGE AND STABILITY
5
5.
MATERIALS SUPPLIED
5
6.
MATERIALS REQUIRED, NOT SUPPLIED
6
7.
LIMITATIONS
8
8.
TECHNICAL HINTS
8
ASSAY PREPARATION
9.
REAGENT PREPARATION
9
10.
CONTROL PREPARATION
11
11.
STANDARD PREPARATION
12
12.
SAMPLE COLLECTION AND STORAGE
14
13.
PLATE PREPARATION
15
ASSAY PROCEDURE
14.
ASSAY PROCEDURE
16
DATA ANALYSIS
15.
CALCULATIONS
18
16.
TYPICAL DATA
19
17.
ASSAY SENSITIVITY
19
18.
ASSAY SPECIFICITY
19
RESOURCES
19.
TROUBLESHOOTING
20
20.
NOTES
22
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1
INTRODUCTION
1. BACKGROUND
Abcam’s Estrogen Receptor ELISA kit is an in vitro ELISA (EnzymeLinked Immunosorbent Assay) designed for accurate quantitative
measurement of Human, mouse and hamster Estrogen Receptor
Alpha concentrations in cell and tissue nuclear extracts.
The Estrogen Receptor Alpha ELISA kit simplifies the measurement of
ERα contained in cell and tissue samples by using the “Sandwich
ELISA” method for detecting a protein. This method uses two
antibodies that each recognize a distinct epitope on the protein of
interest. The kit provides an ELISA plate that is coated with the first
antibody, called the Capture Antibody, which is used to capture the
protein from the sample. The second antibody, called the Detecting
Antibody, is used to detect the protein bound by the Capture Antibody.
An HRP-conjugated Secondary Antibody is then used to quantitate the
amount of bound Detecting Antibody. Subsequent incubation with
developing solution provides an easily quantified colorimetric readout.
Estrogen receptor alpha (ERα) belongs to the nuclear receptor (NR)
superfamily of structurally related ligand-inducible transcription factors.
NRs act in combination with other transcription factors to regulate the
expression of gene networks involved in cell growth and development,
apoptosis, homeostasis, inflammation, lipid metabolism, the
reproductive cycle and other fundamental biological processes. ERα is
also a well-established marker of breast cancer hormone sensitivity,
and the quantification of estrogen receptors in breast tumors has been
routinely performed in clinical laboratories to aid in the selection
between hormonal and chemotherapy and also to predict prognosis.
Because of ERα’s critical role in cell biology, it is important to measure
the total amounts of ERα contained in different cell types and tissues.
Traditional methods for monitoring ERα protein levels, such as
Western blotting, EMSA, immunohistochemistry (IHC) and reporter
gene assays, are time consuming and not suitable to high-throughput
applications.
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2
INTRODUCTION
ER-regulated genes are involved in a variety of cellular pathways that
are currently being deciphered by academic and pharmaceutical
laboratories for new target discovery. However, there is a lack of
standardized assays that measure cellular levels of ER.
Once the samples are prepared, this assay is completed in less than
4 hours. As this assay is performed
in 96-well plates, a large number of
samples
can
be
handled
simultaneously,
enabling
highthroughput automation. This assay is
specific for ERα and can be used to
detect ERα in as little as 0.6 µg of
nuclear extract from MCF-7 cells.
The Estrogen Receptor Alpha ELISA
Kit has many applications including
the study of ER transcriptional
activity regulation and protein
Structure / function studies of ER and
its mutated variants in areas such as
osteoporosis, arteriosclerosis and
breast cancer.
2. ASSAY SUMMARY
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3
INTRODUCTION
Prepare all reagents, samples, and controls as instructed. Plate is
supplied pre-coated with capture antibody.
Add sample to appropriate wells. Incubate at room temperature.
Add primary detection antibody. Incubate at room temperature.
Aspirate and wash each well. Add HRP conjugated secondary
antibody, which binds the primary antibody. Incubate at room
temperature.
Aspirate and wash each well. Add developing solution until color
develops and then add the stop solution. Immediately begin recording
the
color
development.
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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 components at conditions advised 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. Reagent Preparation.
5. MATERIALS SUPPLIED
1 x 13 μL
Storage
Condition
(Before
Preparation)
+2-8°C
HRP-conjugated antibody
6 μL (0.2 µg/µL)
+2-8°C
MCF-7 nuclear extract
40 μL (2.5 µg/µL)
-80°C
Diluent Buffer
1 x 22 mL
-20°C
10X Wash Buffer
1 x 22 mL
+2-8°C
Developing Solution
1 x 11 mL
+2-8°C
Stop Solution
Pre-Coated 96 Well Microplate
(12 x 8 well strips)
Plate sealer
1 x 11 mL
+2-8°C
96 wells
+2-8°C
1 x 1 unit
Room temperature
Item
Amount
ERα detecting antibody
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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:

Multi-channel pipettor

Multi-channel pipettor reservoirs

Shaking platform

Microplate spectrophotometer capable of reading at 450 nm
(655 nm as optional reference wavelength)
These materials are not included in the kit, but will be required for
quantification via the production of a standard curve (section 12.
Sample Collection and Storage):

Recombinant Estrogen Receptor alpha protein
These materials are not included in the kit, but will be required for the
suggested nuclear extraction protocol (section 11. Standard
Preparation):

Phosphate Buffered Saline (PBS)
10X PBS
For 250 mL, mix:
0.1 M phosphate buffer, pH 7.5
3.55 g Na2HPO4 +
0.61 g KH2PO4
1.5 M NaCl
21.9 g
27 mM KCl
0.5 g

Adjust to 250 mL with distilled water. Prepare a 1X PBS solution
by adding 10 mL 10X PBS to 90 mL distilled water. Sterilize the
1X PBS by filtering through a 0.2 μm filter. The 1X PBS is at
pH 7.5. Store the filter-sterilized 1X PBS solution at 4°C.

PIB (Phosphatase Inhibitor Buffer)
1X PIB
mix:
For
25 mM NaF
52 mg
250 mM β-glycerophosphate
0.55 g
250 mM para-nitrophenyl phosphate (PNPP)
1.15 g
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10
mL,
6
GENERAL INFORMATION
25 mM NaVO3
31 mg
Adjust to 10 mL with distilled water. Mix the chemicals by
vortexing. Incubate the solution at 50ºC for 5 minutes. Mix again.
Store at -20°C.

PBS/PIB
Prior to use, add 500 μL of PIB into 10 mL of 1X PBS.

HB (Hypotonic Buffer)
1X HB
For 50 mL, mix:
20 mM Hepes, pH 7.5
0.24 g
5 mM NaF
12 mg
10 μM Na2MoO4
5 μL of a 0.1 M solution
0.1 mM EDTA
10 μL of a 0.5 M solution
Adjust pH to 7.5 with 1 N NaOH. Adjust volume to 50 mL with
distilled water. Sterilize by filtering through a 0.2 μm filter. Store
the filter-sterilized solution at 4°C.

Lysis Buffer
1X Lysis Buffer
For 50 mL, mix:
20 mM Hepes, pH 7.5
0.24 g
400 mM NaCl
1.17 g
0.1 mM EDTA
1.5 mg
10 mM NaF
21 mg
10 µM Na2MoO4
0.12 mg
1 mM NaVO3
6.1 mg
20% glycerol
10 mL
10 mM PNPP
0.23 g
10 mM beta-glycerophosphate
0.11 g
Adjust pH to 7.5 with 1 N NaOH. Adjust volume to 50 mL with
distilled water. Store at 4°C. Just before use, make up Complete
Lysis Buffer by adding 1 µL of 1 M DTT and 10 µL of Protease
Inhibitor Cocktail per mL of Lysis Buffer.
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7
GENERAL INFORMATION
7. LIMITATIONS

Assay kit intended for research use only. Not for use in diagnostic
procedures

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
8. TECHNICAL HINTS

Samples generating values higher than the highest standard
should be further diluted in the appropriate sample dilution buffers

Avoid foaming or bubbles when mixing or reconstituting
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

The Stop Solution is corrosive. Wear personal protective
equipment when handling, i.e. safety glasses, gloves and labcoat.

Complete removal of all solutions and buffers during wash steps.

This kit is sold based on number of tests. A ‘test’ simply
refers to a single assay well. The number of wells that
contain sample, control or standard will vary by product.
Review the protocol completely to confirm this kit meets your
requirements. Please contact our Technical Support staff
with any questions.
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8
ASSAY PREPARATION
9. REAGENT PREPARATION
Equilibrate all reagents and samples to room temperature (18-25°C)
prior to use.
9.1
1X Wash Buffer
Prepare the amount of 1X Wash Buffer required for the
assay as follows: For every 10 mL of 1X Wash Buffer
required, dilute 1 mL 10X Wash Buffer with 9 mL distilled
water. Mix gently to avoid foaming.
The 1X Wash Buffer may be stored at 4°C for one week.
The Tween 20 contained in the 10X Wash Buffer may form
clumps, therefore homogenize the buffer by vortexing for
2 minutes prior to use.
9.2
Antibody Binding Buffer
Dilute the ERα detecting antibody to 1:400 and HRPconjugated secondary antibody to 1:1,000 with the Diluent.
Use 50 µL of diluted antibody per well.
Depending on the particular assay, the signal: noise ratio
may be optimized by using higher dilutions of both
antibodies. This may decrease the sensitivity of the assay.
9.3
Developing Solution
The Developing Solution must be warmed to room
temperature for at least 1 hour before use. Prior to use,
transfer the amount of Developing Solution required for the
assay into a secondary container, avoiding direct exposure
to intense light. After use, discard any remaining solution
that was transferred into the secondary container.
This solution is light sensitive, therefore, we recommend
avoiding direct exposure to intense light during storage. The
Developing Solution may develop a yellow hue over time.
This does not affect product performance. A blue color
present in the solution indicates that it has been
contaminated and must be discarded.
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9
ASSAY PREPARATION
9.4
Stop Solution
Prior to use, transfer the amount of Stop Solution required
for the assay into a secondary container. After use, discard
remaining Stop Solution.
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10
ASSAY PREPARATION
10. CONTROL PREPARATION
Positive control (MCF-7 Nuclear extract)
The MCF-7 nuclear extract is provided as a positive control to
ensure that the kit reagents are functional. Sufficient extract is
provided for 20 reactions. This extract is optimized to give a
strong signal when used at 5 µg/well.
We recommend aliquoting the extract in 5 µL fractions and storing
at -80ºC. Avoid multiple freeze/thaw cycles of the extract.
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11
ASSAY PREPARATION
11. STANDARD PREPARATION

For optional quantification of the amount of Estrogen Receptor
alpha please use the following protocol.

The Estrogen receptor alpha protein standard must be
reconstituted with the appropriate volume of Diluent Buffer
immediately prior to use.
11.1 Reconstitute the Estrogen Receptor alpha standard sample
by adding an appropriate volume of Diluent Buffer to
produce a 100 ng/μL working stock of recombinant protein.
Mix thoroughly and gently.
11.2 Make up a 2.0 ng/µL solution by adding 2.4 µL of the
100 ng/µL working stock to 117.6 µL of Diluent Buffer. This
is the 2,000 pg/mL Standard #1 Solution (see table below).
Note: The reconstituted Standard #1 should be discarded
after use and not stored for reuse.
11.3 Label six tubes with Standards #2 – 8.
11.4 Add 60 μL Diluent Buffer into tubes #2 – 8.
11.5 Prepare Standard #2 by transferring 300 μL
Standard #1 to tube #2. Mix thoroughly and gently.
from
11.6 Prepare Standard #3 by transferring 300 μL from Standard
#2 to tube #3. Mix thoroughly and gently.
11.7 Using the table below as a guide, repeat for tubes #4
through to tube #7.
11.8 Standard #8 contains no protein and is the Blank control.
11.9 50 µL from each tube will be aliquoted to the wells in Step
15.1 of the Assay Procedure (section 15) and will
correspond to the following quantities of ERα: 100, 50, 25,
12.5, 6.25, 3.125, 1.5625 and 0.0 ng/well.
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12
ASSAY PREPARATION
Standard
#
1
2
3
4
5
6
7
8 (Blank)
Sample to
Dilute
Standard #1
Standard #2
Standard #3
Standard #4
Standard #5
Standard #6
Diluent
Buffer
Volume to Volume of
Dilute
Diluent
(µL)
(µL)
See Step 11.1
60
60
60
60
60
60
60
60
60
60
60
60
N/A
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60
Starting
Conc.
(pg/mL)
2,000
1,000
500
250
125
62.5
Final
Conc.
(pg/mL)
2,000
1,000
500
250
125
62.5
31.2
0
0
13
ASSAY PREPARATION
12. SAMPLE COLLECTION AND STORAGE
Preparation of Nuclear Extract (suggested protocol)

For reagent preparation for this protocol see section 6.
Materials Required, not Supplied.

This procedure can be used for a confluent cell layer of 75
cm2 (100 mm dish). The yield is approximately 0.5 mg of
nuclear proteins for 107 cells.
12.1 Wash cells with 10 mL of ice-cold PBS/PIB.
12.2 Add 10 mL of ice-cold PBS/PIB and scrape the cells off the
dish with a cell lifter. Transfer the cells into a pre-chilled
15 mL tube and spin at 300 x g for 5 minutes at 4°C.
12.3 Resuspend the pellet in 1 mL of ice-cold HB buffer by gentle
pipetting and transfer the cells into a pre-chilled 1.5 mL tube.
12.4 Allow the cells to swell on ice for 15 minutes.
12.5 Add 50 µL 10% Nonidet P-40 (0.5% final) and mix by gentle
pipetting.
12.6 Centrifuge the homogenate for 30 seconds at 4°C in a
microcentrifuge.
12.7 Discard the supernatant (which contains the cytoplasm and
RNA) carefully without disturbing the pellet. Resuspend the
nuclear pellet in 50 µL Complete Lysis Buffer and rock the
tube gently on ice for 30 minutes on a shaking platform.
12.8 Centrifuge for 10 minutes at 14,000 x g at 4°C and save the
supernatant (nuclear extract).
12.9 Determine the protein concentration of the extract by using a
Bradford-based assay.
Storage of the nuclear extract: Aliquot and store at -80°C. Avoid
freeze/thaw cycles.
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14
ASSAY PREPARATION
13. 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.

For each assay performed, a minimum of 2 wells must be used as
blanks, omitting primary antibody from well additions.

For statistical reasons, we recommend each sample, control and
blank should be assayed with a minimum of two replicates
(duplicates).

Well effects have not been observed with this assay.

If less than 8 wells in a strip are required, cover the unused wells
with a portion of the plate sealer while you perform the assay.

The content of these wells is stable at room temperature if kept
dry and, therefore, can be used later for a separate assay. Store
the unused strips in the aluminum pouch at 4°C.

Use the strip holder for the assay.
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15
ASSAY PROCEDURE
14. 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.

Prepare all reagents, working standards, and samples as
directed in the previous sections.
Binding of ERα to the capture antibody
14.1 Sample wells: Add 50 µL of sample diluted in Diluent Buffer
to each well to be used. We recommend using 2.5 to 50 µg
of nuclear extract diluted in Diluent Buffer per well. A
protocol for preparing nuclear extracts is provided below.
Control wells: Add 5 µg of the provided MCF-7 nuclear
extract diluted in 50 µL of Diluent Buffer to each well to be
used (2 µL of extract in 48 µL of Diluent Buffer per well).
Blank wells: Add 50 µL Diluent Buffer only per well.
OPTIONAL – Protein standard wells: Add 50 µL of the
appropriate protein standard diluted in Diluent Buffer to each
well being used.
14.2 Use the provided adhesive cover to seal the plate. Incubate
for 1 hour at room temperature with mild agitation (100 rpm
on a rocking platform).
14.3 Wash each well 3 times with 200 μL 1X Washing Buffer. For
each wash, flick the plate over a sink to empty the wells,
then tap the inverted plate 3 times on absorbent paper
towels.
Binding of primary antibody
14.4 Add 50 µL diluted ERα antibody (1:400 dilution in Diluent
Buffer) to all wells being used.
14.5 Cover the plate and incubate for 1 hour at room temperature
with gentle rocking.
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16
ASSAY PROCEDURE
14.6 Wash the wells 3 times with 200 μL 1X Wash Buffer (as
described in Step 14.3).
Binding of secondary antibody
14.7 Add 50 µL of diluted HRP-conjugated antibody (1:1000
dilution in Diluent Buffer) to all wells being used.
14.8 Cover the plate and incubate for 1 hour at room temperature
with gentle rocking.
14.9 During this incubation, place the Developing Solution at
room temperature.
14.10 Wash the wells 4 times with 200 μL 1X Wash Buffer (as
described in Step 14.3).
Colorimetric reaction
14.11 Transfer the amount of Developing Solution required for the
assay into a secondary container. Add 100 µL Developing
Solution to all wells being used.
14.12 Incubate 2-10 minutes at room temperature protected from
direct light. Monitor the blue color development in the
sample and positive control wells until it turns medium to
dark blue. Blank wells should remain faint to light blue. Do
not overdevelop.
14.13 Add 100 μL Stop Solution. In presence of the acid, the blue
color turns yellow.
14.14 Read absorbance on a spectrophotometer within 5 minutes
at 450 nm with a reference wavelength of 655 nm. Blank the
plate reader according to the manufacturer’s instructions
using the blank wells.
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17
DATA ANALYSIS
15. CALCULATIONS
If you have generated a standard curve using a recombinant ER
protein, average the duplicate readings for each standard, control, and
sample and subtract the optical density (OD) obtained from the zero
standard.
Plot the OD for the standards against the quantity (ng/well) of the
standards and draw the best fit curve. The data can be linearized using
log/log paper and regression analysis may also be applied.
To quantify the amount of ER in the samples, find the absorbance
value for the samples on the y-axis and extend a horizontal line to the
standard curve. At the intersection point extend a vertical line to the xaxis and read the corresponding standard value. Note: If the samples
have been diluted, the value read from the standard curve must be
multiplied by the dilution factor.
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18
DATA ANALYSIS
16. TYPICAL DATA
This data is provided for demonstration purposes only.
Figure 1: Different amounts of nuclear extracts from MCF-7 and MDA-MB-231
cells were analyzed for cellular levels of ERα using the ERα ELISA Kit.
17. ASSAY SENSITIVITY
Sensitivity: > 0.6 μg nuclear extract/well.
Range of detection: Estrogen Receptor Alpha ELISA kit provides
quantitative results from 0.6 to 10 µg of nuclear extract/well.
18. ASSAY SPECIFICITY
Cross-reactivity: Estrogen Receptor Alpha ELISA Kit detects ERα
from Human, mouse and hamster origin. This assay is not
recommended for use with samples from rat origin. Cross-reactivity
with other species has not been determined.
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19
RESOURCES
19. TROUBLESHOOTING
Problem
No signal or
weak signal
in all wells
Cause
Omission of key reagent
Substrate or conjugate is no
longer active
Enzyme inhibitor present
Plate reader settings not
optimal
Incorrect assay temperature
Inadequate volume of
Developing Solution
Developing time too short
High
background
in all wells
Developing time too long
Concentration of antibodies
too high
Inadequate washing
Uneven color
development
Incomplete washing of wells
Well cross-contamination
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Solution
Check that all reagents have
been added in the correct
order
Test conjugate and substrate
for activity
Sodium azide will inhibit the
peroxidise reaction, follow our
recommendations to prepare
buffers
Verify the wavelength and filter
settings in the plate reader
Bring substrate to room
temperature
Check to make sure that
correct volume is delivered by
pipette
Increase the development time
up to 30 minutes
Stop enzymatic reaction as
soon as the positive wells turn
medium-dark blue
Increase antibody dilutions
Ensure all wells are filled with
Washing Buffer and follow
washing recommendations
Ensure all wells are filled with
Washing Buffer and follow
washing recommendations
Follow washing
recommendations
20
RESOURCES
Problem
High
background
in sample
wells
Cause
Too much sample per well
Concentration of antibodies
too high
No signal or
weak signal
in sample
wells
Not enough ERα in the sample
added per well
Solution
Decrease amount of sample
Perform antibody titration to
determine optimal working
concentration. Start using
1:800 for primary antibody.
The sensitivity of the assay will
be decreased
Increase amount of sample
ERα is poorly expressed or
inactivated in samples
Check ERα expression in the
studied sample
Samples are not from correct
origin
Refer to cross-reactivity
information
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RESOURCES
20. NOTES
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22
RESOURCES
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RESOURCES
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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
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Email: supportscientifique@abcam.com | Tel: 01-46-94-62-96
Germany
Email: wissenschaftlicherdienst@abcam.com | Tel: 030-896-779-154
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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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
25