ab65336
Triglyceride
Quantification Assay kit
(Colorimetric/Fluorometric)
Instructions for Use
For the rapid, sensitive and accurate measurement of Triglyceride in
various samples.
This product is for research use only and is not intended for diagnostic
use.
Version 6 Last Updated 27 March 2015
Table of Contents
INTRODUCTION
1.
2.
BACKGROUND
ASSAY SUMMARY
2
3
GENERAL INFORMATION
3.
4.
5.
6.
7.
8.
PRECAUTIONS
STORAGE AND STABILITY
MATERIALS SUPPLIED
MATERIALS REQUIRED, NOT SUPPLIED
LIMITATIONS
TECHNICAL HINTS
4
4
5
5
6
7
ASSAY PREPARATION
9.
10.
11.
REAGENT PREPARATION
STANDARD PREPARATION
SAMPLE PREPARATION
8
10
12
ASSAY PROCEDURE and DETECTION
12.
ASSAY PROCEDURE and DETECTION
14
DATA ANALYSIS
13.
14.
CALCULATIONS
TYPICAL DATA
16
18
RESOURCES
15.
16.
17.
18.
19.
QUICK ASSAY PROCEDURE
TROUBLESHOOTING
FAQ
INTERFERENCES
NOTES
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20
21
23
25
26
1
INTRODUCTION
1.
BACKGROUND
Triglyceride Quantification Assay Kit (Colorimetric/Fluorometric)
(ab65336) provides a sensitive, easy assay to measure TG
concentration in a variety of samples. In the assay, TG are converted
to free fatty acids and glycerol. The glycerol is then oxidized to
generate a product which reacts with the probe to generate color
(spectrophotometry at λ = 570 nm) and fluorescence (Ex/Em =
535/587 nm).
The kit can detect 2 pmol-10 nmol (or 2 – 10000 pM range) of
triglyceride in various samples.
The kit also detects monoglycerides and diglycerides.
Triglycerides (TG) are the main constituent of vegetable oil, animal fat,
LDL and VLDL, and play an important role as transporters of fatty
acids as well as serving as an energy source. TG are broken down into
fatty acids and glycerol, after which both can serve as substrates for
energy producing and metabolic pathways. High blood levels of TG are
implicated in atherosclerosis, heart disease and stroke as well as in
pancreatitis.
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2
INTRODUCTION
2. ASSAY SUMMARY
Standard curve preparation
Sample preparation
Add lipase and incubate at RT for 20 min
Add reaction mix and incubate at RT for 60 min
protected from light
Measure optical density (OD570 nm) or
fluorescence (Ex/Em = 535/590 nm)
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3
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 -20ºC in the dark immediately upon receipt. Kit has a
storage time of 1 year from receipt, providing components have
not been reconstituted.
Refer to list of materials supplied for storage conditions of individual
components. Observe the storage conditions for individual prepared
components in section 5.
Aliquot components in working volumes before storing at the
recommended temperature. Reconstituted components are stable
for 2 months.
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4
GENERAL INFORMATION
5. MATERIALS SUPPLIED
Triglyceride Assay Buffer
25 mL
Storage
Condition
(Before
Preparation)
-20°C
Triglyceride Probe (in DMSO,
anhydrous)
200 μL
-20°C
-20°C
Lipase (Lyophilized)
1 vial
-20°C
-20°C
Triglyceride Enzyme Mix
(Lyophilized)
1 vial
-20°C
-20°C
0.3 mL
-20°C
-20°C
Item
1 mM Triglyceride Standard
Amount
Storage
Condition
(After
Preparation)
-20°C
6. MATERIALS REQUIRED, NOT SUPPLIED
These materials are not included in the kit, but will be required to
successfully utilize this assay:

MilliQ water or other type of double distilled water (ddH2O)

Microcentrifuge

Pipettes and pipette tips

Colorimetric or fluorescent microplate reader – equipped with filter
for OD570 nm or Ex/Em = 535/587 nm (respectively)

96 well plate: black plates (clear bottoms) for fluorometric assay;
clear plates for colorimetric assay

Orbital shaker

Heat block or water bath

Vortex

Dounce homogenizer or pestle (if using tissue)

NP-40
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5
GENERAL INFORMATION
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

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.

Keep enzymes, heat labile components and samples on ice during
the assay.

Make sure all buffers and solutions are at room temperature before
starting the experiment.

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.

Make sure you have the right type of plate for your detection
method of choice.

Make sure the heat block/water bath and microplate reader are
switched on.
or
bubbles
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when
mixing
or
reconstituting
7
ASSAY PREPARATION
9. REAGENT PREPARATION

Briefly centrifuge small vials at low speed prior to opening.
9.1
Triglyceride Assay Buffer:
Ready to use as supplied. Equilibrate to room temperature
before use. Store at -20°C.
9.2
Triglyceride Standard:
Frozen storage may cause the triglyceride standard to
separate from the aqueous phase. To re-dissolve, keep the
cap tightly closed and place in a hot water bath (~80-100°C)
for 1 min or until the standard looks cloudy, and then vortex
for 30 sec, the standard should become clear. Repeat the
heat and vortex one more time. The Triglyceride Standard is
now completely in solution, and ready to use. Aliquot
standard so that you have enough volume to perform the
desired number of assays. Store aliquots at - 20°C.
9.3
Triglyceride Probe:
Ready to use as supplied. Warm by placing in a 37°C bath
for 1 – 5 minutes to thaw the DMSO solution before use.
NOTE: DMSO tends to be solid when stored at -20°C,
even when let at room temperature, so it needs to melt
for few minutes at 37°C. Aliquot probe so that you have
enough volume to perform the desired number of assays.
Store at - 20°C protected from light and moisture. Once the
probe is thawed, use within two months.
9.4
Triglyceride Enzyme mix:
Reconstitute in 220 μL Triglyceride Assay Buffer. Keep on
ice during the assay. Aliquot enzyme mix so that you have
enough volume to perform the desired number of assays.
Store aliquots at - 20°C. Use within two months.
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8
ASSAY PRE
ASSAY PREPARATION
9.5
Lipase:
Reconstitute in 220 μL Triglyceride Assay Buffer. Keep on
ice during the assay. Aliquot lipase so that you have enough
volume to perform the desired number of assays. Store at 20°C. Use within two months.
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9
ASSAY PRE
ASSAY PREPARATION
10.STANDARD PREPARATION

Always prepare a fresh set of standards for every use.

Diluted standard solution is unstable and must be used within 4
hours.
10.1
For the colorimetric assay
10.1.1 Prepare a 0.2 mM Triglyceride standard by diluting 100 µL
of the 1 mM standard in 400 µL of Assay Buffer.
10.1.2 Using 0.2 mM Triglyceride standard, prepare standard
curve dilution as described in the table in a microplate or
microcentrifuge tubes:
Standard
#
1
2
3
4
5
6
Volume of
Triglyceride
Standard
(µL)
0
30
60
90
120
150
Assay
Buffer
(µL)
150
120
90
60
30
0
Final
volume
in well
(µL)
50
50
50
50
50
50
End
[Triglyceride] in
well
0 nmol/well
2 nmol/well
4 nmol/well
6 nmol/wZell
8 nmol/well
10 nmol/well
Each dilution has enough amount of standard to set up duplicate
reading (2 x 50 µL).
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10
ASSAY PRE
ASSAY PREPARATION
10.2
For the flurometric assay:
10.2.1 Prepare a 0.2 mM Triglyceride standard by diluting 40 µl of
the 1mM standard in 160 µL of Assay Buffer.
10.2.2 Prepare a 0.02 mM Triglyceride standard by diluting 50 µL
of the 0.2mM Triglyceride standard with 450 µL of ddH2O.
10.2.3 Using 0.02 mM Triglyceride standard, prepare standard
curve dilution as described in the table in a microplate or
microcentrifuge tubes:
Standard
#
1
2
3
4
5
6
Volume of
Triglyceride
Standard
(µL)
0
30
60
90
120
150
Assay
Buffer
(µL)
150
120
90
60
30
0
Final
volume
in well
(µL)
50
50
50
50
50
50
End
[Triglyceride] in
well
0 pmol/well
200 pmol/well
400 pmol/well
600 pmol/well
800 pmol/well
1 nmol/well
Each dilution has enough amount of standard to set up duplicate
reading (2 x 50 µL).
NOTE: If your sample readings fall out the range of your
fluorometric standard curve, you might need to adjust the dilutions
and create a new standard curve.
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11
ASSAY PRE
ASSAY PREPARATION
11.SAMPLE PREPARATION
General Sample information:

We recommend performing several dilutions of your sample to
ensure the readings are within the standard value range.

We recommend that you use fresh samples. If you cannot perform
the assay at the same time, we suggest that you complete the
Sample Preparation step before storing the samples. Alternatively,
if that is not possible, we suggest that you snap freeze cells or
tissue in liquid nitrogen upon extraction and store the samples
immediately at -80°C. When you are ready to test your samples,
thaw them on ice. Be aware however that this might affect the
stability of your samples and the readings can be lower than
expected.
11.1
Cells (adherent or suspension) samples:
11.1.1 Harvest the amount of cells necessary for each assay (initial
recommendation = 1 x 107 cells).
11.1.2 Wash cells with cold PBS.
11.1.3 Resuspend and homogenize samples in 1 mL of 5% NP40/ddH2O solution.
11.1.4 Slowly heat the samples to 80 – 100°C in a water bath for
2 – 5 minutes or until the NP-40 becomes cloudy, then cool
down to room temperature.
11.1.5 Repeat previous step to solubilize all triglyceride.
11.1.6 Centrifuge for 2 minutes at top speed using
microcentrifuge to remove any insoluble material.
a
11.1.7 Dilute samples 10-fold with ddH2O before proceeding with
the assay.
11.2
Tissue Samples:
11.2.1 Harvest the amount of tissue necessary for each assay
(initial recommendation = 100 mg tissue).
11.2.2 Wash tissue in cold PBS.
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12
ASSAY PRE
ASSAY PREPARATION
11.2.3 Resuspend and homogenize in 1 mL of 5%NP-40/ddH2O
solution using a Dounce homogenizer or pestle with 10 – 15
passes.
11.2.4 Slowly heat the samples to 80 – 100°C in a water bath for
2 – 5 minutes or until the NP-40 becomes cloudy, then cool
down to room temperature.
11.2.5 Repeat the heating one more time to solubilize all
triglyceride.
11.2.6 Centrifuge for 2 minutes at top speed using
microcentrifuge to remove any insoluble material.
a
11.2.7 Dilute samples 10-fold with ddH2O before proceeding with
the assay.
11.3 Serum samples:
Serum contains 0.1 – 6 mM triglyceride, which can be tested
directly.
NOTE: We suggest using different volumes of sample to
ensure readings are within the Standard Curve range.
Endogenous compounds in the sample may interfere with the
reaction. To ensure accurate determinations of Triglyceride in
our sample, we recommend spiking samples with a known
amount of Standard (2 – 10 nmol).
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13
ASSAY PROCEDURE and DETECTION
12. ASSAY PROCEDURE and DETECTION
●
Equilibrate all materials and prepared reagents to room
temperature prior to use.
●
It is recommended to assay all standards, controls and
samples in duplicate.
12.1
Set up Reaction wells:
-
Standard wells = 50 µL standard dilutions.
-
Sample wells = 2 – 50 µL samples (adjust volume to 50 µL/well
with Triglyceride Assay Buffer).
-
Sample background control wells = 2 – 50 µL samples (adjust
volume to 50 µL/well with Triglyceride Assay Buffer). NOTE: for
samples that contain glycerol, as it can interfere with the lipase
activity.
12.2
Add Lipase:
Add lipase to reactions as follows:
-
Standard wells = 2 µL Lipase.
-
Sample wells = 2 µL Lipase
-
Sample background wells = 2 µL Triglyceride Assay Buffer (do
not add Lipase to these samples).
Mix and incubate 20 minutes at room temperature to convert
triglyceride to glycerol and fatty acid.
12.3
Triglyceride Reaction Mix:
Prepare 50 µL of Reaction Mix for each reaction:
Component
Colorimetric
Reaction
(µL)
Fluorometric
Mix
Reaction
Triglyceride Assay buffer
46
47.6
Triglyceride Probe*
2
0.4
Triglyceride Enzyme Mix
2
2
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Mix
(µL)
14
ASSAY PRE
ASSAY PREPARATION
*For fluorometric readings, using 0.4 μL/well of the Triglyceride
probe decreases the background readings, therefore
increasing detection sensitivity.
Mix enough reagents for the number of assays (samples,
standards and background control) to be performed. Prepare a
Master Mix of the Reaction Mix to ensure consistency. We
recommend the following calculation:
X µL component x (Number samples + standards +
background control + 1)
12.4
Add 50 µL of Reaction Mix into each well.
12.5
Mix and incubate at room temperature for 60 minutes
protected from light.
12.6
Measure assay in a suitable microplate reader:
-
Colorimetric assay: measure OD570nm
-
Fluorometric assay: measure Ex/Em = 535/590 nm.
The reaction is stable for at least two hours.
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15
DATA ANALYSIS
13. CALCULATIONS

Samples producing signals greater than that of the highest
standard should be further diluted in appropriate buffer and
reanalyzed, then multiplying the concentration found by the
appropriate dilution factor.

For statistical reasons, we recommend each sample should be
assayed with a minimum of two replicates (duplicates).
13.1
Average the duplicate reading for each standard and sample.
13.2 If sample background control is significant, then subtract the
sample background control from the sample readings.
13.3 Subtract the mean absorbance value of the blank (Standard
#1) from all standard and sample readings. This is the corrected
absorbance.
13.4 Plot the corrected absorbance values for each standard as a
function of the final concentration of Triglyceride.
13.5 Draw the best smooth curve through these points to construct
the standard curve. Most plate reader software or Excel can plot
these values and curve fit. Calculate the trendline equation based
on your standard curve data (use the equation that provides the
most accurate fit).
13.6 Extrapolate sample readings from the standard curve plotted
using the following equation:
‒ (𝑦 ‒ 𝑖𝑛𝑡𝑒𝑟𝑐𝑒𝑝𝑡)
𝑇𝑠 = 𝐶𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒
𝑆𝑙𝑜𝑝𝑒
(
13.7
)
Concentration of samples in the test samples is calculated as:
𝑇𝑠 ∗ 𝐷
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 = 𝑆𝑣
( )
Where:
Ts = amount of Triglyceride (nmol) from standard curve.
Sv = volume of sample (µL) added in sample wells.
D = sample dilution factor.
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16
ASSAY PRE
ASSAY PREPARATION
Triglyceride molecular weight = 885.4 g/mol
13.8
For spiked samples, correct any sample interference by
subtracting the sample from spiked sample reading.
For spiked samples, the concentration of Triglyceride in
sample well is calculated as:
(𝑂𝐷𝑠 𝑐𝑜𝑟)
𝑆𝑎 = (𝑂𝐷𝑠 + 𝑆𝑎
𝑐𝑜𝑟) ‒ (𝑂𝐷𝑠 𝑐𝑜𝑟) ∗ 𝑇𝐺𝐴 𝑆𝑝𝑖𝑘𝑒 (𝑛𝑚𝑜𝑙)
(
)
Where:
ODs cor = OD sample corrected.
ODs = OD sample.
Sa cor = TGA (triglyceride) amount from standard curve
corrected.
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17
ASSAY PRE
ASSAY PREPARATION
14. TYPICAL DATA
TYPICAL STANDARD CURVE – Data provided for demonstration
purposes only. A new standard curve must be generated for each
assay performed.
Figure 1: Typical Triglyceride Standard calibration curve using colorimetric
reading.
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18
ASSAY PRE
ASSAY PREPARATION
Figure 2: Determination of Triglyceride in pooled normal human serum.
Serum sample (3 µL) was spiked with a known amount of Triglyceride as
internal Standard (4 nmol) and assayed according to the kit protocol.
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19
RESOURCES
15.QUICK ASSAY PROCEDURE
NOTE: This procedure is provided as a quick reference for
experienced users. Follow the detailed procedure when performing
the assay for the first time.

Solubilize Triglyceride standard, thaw Triglyceride probe and
prepare enzyme mix and lipase (aliquot if necessary); get
equipment ready.

Prepare appropriate standard curve for your detection method of
choice (colorimetric or fluorometric).

Prepare samples in optimal dilutions so that they fit standard curve
readings.

Set up plate in duplicate for standard (50 µL), samples (50 µL),
and if appropriate, for sample background control wells (50 µL).

Add 2 µL Lipase to standard and sample wells or 2 µL Assay
Buffer to sample background control wells. Mix and incubate for 20
min at RT.

Prepare 50 µL of appropriate Reaction Mix (Number samples +
standards + background control + 1) for each reaction.
Component
Colorimetric
Reaction
Fluorometric
Mix
Reaction Mix (µL)
(µL)
Triglyceride Assay buffer
46
47.6
Triglyceride Probe*
2
0.4
Triglyceride Enzyme Mix
2
2

Add 50 µL Triglyceride Reaction mix to standard, sample and
sample background control wells.

Incubate plate at RT for 60 min

Measure plate at OD570 nm for colorimetric
Ex/Em = 535/590 nm for fluorometric assay.
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assay
20
or
RESOURCES
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21
RESOURCES
16. TROUBLESHOOTING
Problem
Assay not
working
Sample with
erratic
readings
Lower/
Higher
readings in
samples and
Standards
Cause
Solution
Use of ice-cold buffer
Buffers must be at room
temperature
Plate read at
incorrect wavelength
Check the wavelength and filter
settings of instrument
Use of a different 96well plate
Colorimetric: Clear plates
Fluorometric: black wells/clear
bottom plate
Samples not
deproteinized (if
indicated on protocol)
Cells/tissue samples
not homogenized
completely
Samples used after
multiple free/ thaw
cycles
Use of old or
inappropriately stored
samples
Presence of
interfering substance
in the sample
Use PCA precipitation protocol for
deproteinization
Use Dounce homogenizer,
increase number of strokes
Aliquot and freeze samples if
needed to use multiple times
Use fresh samples or store at 80°C (after snap freeze in liquid
nitrogen) till use
Check protocol for interfering
substances; deproteinize samples
Improperly thawed
components
Thaw all components completely
and mix gently before use
Allowing reagents to
sit for extended times
on ice
Always thaw and prepare fresh
reaction mix before use
Incorrect incubation
times or temperatures
Verify correct incubation times
and temperatures in protocol
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RESOURCES
Problem
Standard
readings do
not follow a
linear pattern
Unanticipated
results
Cause
Solution
Pipetting errors in
standard or reaction
mix
Avoid pipetting small volumes
(< 5 µL) and prepare a master mix
whenever possible
Air bubbles formed in
well
Pipette gently against the wall of
the tubes
Standard stock is at
incorrect
concentration
Always refer to dilutions described
in the protocol
Measured at incorrect
wavelength
Check equipment and filter setting
Samples contain
interfering
substances
Sample readings
above/ below the
linear range
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Troubleshoot if it interferes with
the kit
Concentrate/ Dilute sample so it is
within the linear range
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RESOURCES
17. FAQ
What can be done if the lysed cells are not dissolving?
The amount of 5 % NP-40 in water used can be increased. Also, the
temperature can be raised above 80° C to get the particles into
solution, in addition to repeating the heating and cooling for 2 cycles.
What could be the explanation for negative OD values but positive
BODIPY staining in cells?
The kit can detect 2 pmol – 10 nmol (or 2 – 10000 µM range) of
triglyceride. It could be that the BODIPY staining is showing total lipids
in these cells but the amount of triglycerides is low. The fluorometric
version of this assay is 10X more sensitive than the colorimetric one
and could be chosen to see if the readings make sense. Also, it is
crucial to check the instrument settings while measuring the samples.
Use the correct filter for 570nm detection.
How much Triglyceride is there is serum samples?
Typical serum levels of Triglyceride can range between 0.1 – 6 mM,
individual experimental findings may vary.
Are the triglycerides concentrated on the surface layer of the
supernatant or distributed in the whole supernatant after the
boiling step?
It is possible that triglycerides aggregate upon freezing and thawing
while in an aqueous solution and stick to the walls of the tube which
can skew the results. Also, there could be a layer of fat/oil after boiling.
Once the sample is centrifuged, the liquid can be collected in a fresh
tube and thoroughly vortexed so that when the sample is added to a
well, a homogenous solution is pipetted.
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RESOURCES
Can less than 10 million cells be used for this assay?
Less cells can be used, but the yield of triglycerides might be less. The
number of cells depends on the amount of triglycerides in them. If less
cells are used the volume of NP40-water can be scaled down
proportionately.
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25
RESOURCES
18. INTERFERENCES
These chemicals or biological will cause interferences in this assay
causing compromised results or complete failure.
 Sodium azide content above 0.05%
 Phenol red: typically does not interfere but when the color of the
reaction in the well changes due to the amount used, then the red
color can potentially interfere with the assay.
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RESOURCES
19. NOTES
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27
UK, EU and ROW
Email: [email protected] | Tel: +44-(0)1223-696000
Austria
Email: [email protected] | Tel: 019-288-259
France
Email: [email protected] | Tel: 01-46-94-62-96
Germany
Email: [email protected] | Tel: 030-896-779-154
Spain
Email: [email protected] | Tel: 911-146-554
Switzerland
Email: [email protected]
Tel (Deutsch): 0435-016-424 | Tel (Français): 0615-000-530
US and Latin America
Email: [email protected] | Tel: 888-77-ABCAM (22226)
Canada
Email: [email protected] | Tel: 877-749-8807
China and Asia Pacific
Email: [email protected] | Tel: 108008523689 (中國聯通)
Japan
Email: [email protected] | Tel: +81-(0)3-6231-0940
www.abcam.com | www.abcam.cn | www.abcam.co.jp
Copyright © 2015 Abcam, All Rights Reserved. The Abcam logo is a registered trademark.
All information / detail is correct at time of going to print.
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