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 9 Last Updated: 8 February 2016
Table of Contents
INTRODUCTION
1
1.
BACKGROUND
1
2.
ASSAY SUMMARY
2
GENERAL INFORMATION
3
3.
PRECAUTIONS
3
4.
STORAGE AND STABILITY
3
5.
LIMITATIONS
4
6.
MATERIALS SUPPLIED
4
7.
MATERIALS REQUIRED, NOT SUPPLIED
5
8.
TECHNICAL HINTS
6
ASSAY PREPARATION
7
9.
REAGENT PREPARATION
10.
STANDARD PREPARATION
11.
SAMPLE PREPARATION
7
8
10
ASSAY PROCEDURE
12
12.
12
ASSAY PROCEDURE
DATA ANALYSIS
14
13.
CALCULATIONS
14
14.
TYPICAL DATA
16
RESOURCES
18
15.
QUICK ASSAY PROCEDURE
18
16.
TROUBLESHOOTING
19
17.
INTERFERENCES
21
18.
FAQS
21
19.
NOTES
23
INTRODUCTION
INTRODUCTION
1. BACKGROUND
Triglyceride Quantification Assay Kit (Colorimetric/Fluorometric)
(ab65336) is a sensitive and easy-to-use kit to measure triglyceride
concentration in a variety of samples. In this assay, triglycerides 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 (λ =
570 nm) and fluorescence (Ex/Em = 535/587 nm).
This assay can detect 2 pmol – 10 nmol (or 2 µM – 10 mM range) of
triglyceride in various biological 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. Triglycerides 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
triglycerides are implicated in atherosclerosis, heart disease and stroke
as well as in pancreatitis.
ab65336 Triglyceride Quantification Assay Kit (Colorimetric/Fluorometric)
1
INTRODUCTION
2. ASSAY SUMMARY
Standard curve preparation
Sample preparation
Add lipase and incubate at RT for 20 minutes
Add reaction mix and incubate at RT for 60 minutes protect from light
Measure optical density (OD570 nm) or
fluorescence (Ex/Em = 535/587 nm)
ab65336 Triglyceride Quantification Assay Kit (Colorimetric/Fluorometric)
2
GENERAL INFORMATION
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.

We understand that, occasionally, experimental protocols might
need to be modified to meet unique experimental circumstances.
However, we cannot guarantee the performance of the product
outside the conditions detailed in this protocol booklet.

Reagents should be treated as possible mutagens and should be
handle with care and disposed of properly. Please review the Safety
Datasheet (SDS) provided with the product for information on the
specific components.

Observe good laboratory practices. Gloves, lab coat, and protective
eyewear should always be worn. Never pipet by mouth. Do not eat,
drink or smoke in the laboratory areas.

All biological materials should be treated as potentially hazardous
and handled as such. They should be disposed of in accordance
with established safety procedures.
4. STORAGE AND STABILITY
Store kit at -20°C 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 the Materials Supplied section.
Aliquot components in working volumes before storing at the
recommended temperature. Reconstituted components are stable for
2 months.
ab65336 Triglyceride Quantification Assay kit (Colorimetric/Fluorometric)
3
GENERAL INFORMATION
5. 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.
6. MATERIALS SUPPLIED
Item
Triglyceride Assay Buffer
25 mL
Storage
Condition
(Before
Preparation)
-20°C
Triglyceride Probe (in
DMSO, anhydrous)
Lipase (Lyophilized)
200 μL
-20°C
-20°C
1 vial
-20°C
-20°C
1 vial
-20°C
-20°C
300 µL
-20°C
-20°C
Triglyceride Enzyme Mix
(Lyophilized)
1 mM Triglyceride Standard
Amount
ab65336 Triglyceride Quantification Assay kit (Colorimetric/Fluorometric)
Storage
Condition
(After
Preparation)
-20°C
4
GENERAL INFORMATION
7. 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 OD 570 nm
(colorimetric) or fluorescence at Ex/Em = 535/587 nm (fluorometric)

MilliQ water or other type of double distilled water (ddH2O)

Pipettes and pipette tips, including multi-channel pipette

Assorted glassware for the preparation of reagents and buffer
solutions

Tubes for the preparation of reagents and buffer solutions

96 well plate with clear flat bottom (for colorimetric assay) / 96 well
plate with clear flat bottom, preferably black (for fluorometric assay)

Dounce homogenizer (if using tissue)

NP-40
ab65336 Triglyceride Quantification Assay kit (Colorimetric/Fluorometric)
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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.

Selected components in this kit are supplied in surplus amount to
account for additional dilutions, evaporation, or instrumentation
settings where higher volumes are required. They should be
disposed of in accordance with established safety procedures.

Avoid foaming
components.

Avoid cross contamination of samples or reagents by changing tips
between sample, standard and reagent additions.

Ensure all reagents and solutions are at the appropriate temperature
before starting the assay.

Samples which generate values that are greater than the most
concentrated standard should be further diluted in the appropriate
sample dilution buffer.

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 all necessary equipment is switched on and set at the
appropriate temperature.
or
bubbles
when
mixing
or
ab65336 Triglyceride Quantification Assay kit (Colorimetric/Fluorometric)
reconstituting
6
ASSAY PREPARATION
ASSAY PREPARATION
9. REAGENT PREPARATION

9.1.
Briefly centrifuge small vials at low speed prior to opening
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 minute or
until the standard looks cloudy, and then vortex for 30 seconds,
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. NOTE: each aliquot of standard should be
boiled as described above before use.
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.
9.5.
Lipase:
Reconstitute in 220 μL Triglyceride Assay Buffer. Keep on ice
during the assay. Aliquot lipase so that you have enough volume
ab65336 Triglyceride Quantification Assay kit (Colorimetric/Fluorometric)
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ASSAY PREPARATION
to perform the desired number of assays. Store at - 20°C. Use
within two months.
ab65336 Triglyceride Quantification Assay kit (Colorimetric/Fluorometric)
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ASSAY PREPARATION
10. STANDARD PREPARATION

Always prepare a fresh set of standards for every use.

Discard the working standard dilutions after use as they do not store
well.
10.1. For 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
#
Assay
Buffer
(µL)
Final
volume in
well (µL)
End Conc
TG in well
(nmol/well)
1
Volume of
TG
Standard
(µL)
0
150
50
0
2
30
120
50
2
3
60
90
50
4
4
90
60
50
6
5
120
30
50
8
6
150
0
50
10
Each dilution has enough amount of standard to set up duplicate reading
(2 x 50 µL).
ab65336 Triglyceride Quantification Assay kit (Colorimetric/Fluorometric)
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ASSAY PREPARATION
10.2. For fluorometric 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 Assay buffer.
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
#
Assay
Buffer
(µL)
1
Volume of
Triglyceride
Standard
(µL)
0
End Conc TG in
well (pmol/well)
150
Final
volume
in well
(µL)
50
2
3
30
120
50
200
60
90
50
400
4
90
60
50
600
5
120
30
50
800
6
150
0
50
1000
0
Each dilution has enough amount of standard to set up duplicate reading
(2 x 50 µL).
ab65336 Triglyceride Quantification Assay kit (Colorimetric/Fluorometric)
10
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 your samples
in liquid nitrogen upon extraction and store them 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 a microcentrifuge
to remove any insoluble material.
11.1.7. Dilute samples 10-fold with ddH2O before proceeding with the
assay.
ab65336 Triglyceride Quantification Assay kit (Colorimetric/Fluorometric)
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ASSAY PREPARATION
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.
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 a microcentrifuge
to remove any insoluble material.
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).
ab65336 Triglyceride Quantification Assay kit (Colorimetric/Fluorometric)
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ASSAY PROCEDURE
ASSAY PROCEDURE
12. ASSAY PROCEDURE

Equilibrate all materials and prepared reagents to room
temperature prior to use.

We recommend that you assay all standards, controls and
samples in duplicate.

Prepare all reagents, working standards, and samples as
directed in the previous sections.
NOTE: glycerol present in cell or tissue extracts can interfere with
the lipase activity and generate background in this assay. If you
suspect your samples contain glycerol, set up Sample Background
Controls.
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).
12.2. Add Lipase:
Add lipase to reactions as follows:
Standard wells = 2 µL Lipase.
Sample wells = 2 µL Lipase
Sample Background control 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. 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 reactions + 1)
ab65336 Triglyceride Quantification Assay kit (Colorimetric/Fluorometric)
13
ASSAY PROCEDURE
Colorimetric
Reaction Mix
(µL)
Fluorometric
Reaction Mix
(µL)
Triglyceride Assay buffer
46
47.6
Triglyceride Probe*
2
0.4
Triglyceride Enzyme Mix
2
2
Component
*NOTE: for fluorometric readings, using 0.4 μL/well of the
Triglyceride probe decreases the background readings, therefore
increasing detection sensitivity.
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 output on a microplate reader at OD 570 nm for
colorimetric assay and at Ex/Em = 535/587 nm for a fluorometric
assay.
The reaction is stable for at least two hours.
ab65336 Triglyceride Quantification Assay kit (Colorimetric/Fluorometric)
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DATA ANALYSIS
DATA ANALYSIS
13. CALCULATIONS

Samples producing signals greater than that of the highest standard
should be further diluted in appropriate buffer and reanalyzed, then
multiply the concentration found by the appropriate dilution factor.
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. Concentration of triglyceride in the test samples is calculated as:
𝑇𝑠 ∗ 𝐷
𝑇𝐺 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 = 𝑆𝑣
( )
Where:
Ts = amount of Triglyceride in the sample well calculated from
standard curve (nmol).
Sv = volume of sample added in sample wells (µL).
D = sample dilution factor.
Triglyceride molecular weight = 885.4 g/mol
ab65336 Triglyceride Quantification Assay kit (Colorimetric/Fluorometric)
15
DATA ANALYSIS
13.7. 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) ∗ 𝑇𝐺 𝑆𝑝𝑖𝑘𝑒 (𝑛𝑚𝑜𝑙)
(
)
Where:
ODs cor = OD sample corrected.
ODs = OD sample.
Sa cor = TG (triglyceride) amount from standard curve corrected.
ab65336 Triglyceride Quantification Assay kit (Colorimetric/Fluorometric)
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DATA ANALYSIS
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.
ab65336 Triglyceride Quantification Assay kit (Colorimetric/Fluorometric)
17
DATA ANALYSIS
Figure 2: Fluorometric triglyceride standard curve: mean of duplicates (+/- SD)
with background readings subtracted
Figure 3: Triglyceride measured in cell culture lysates showing quantity (nmol)
per million cells. Samples with the concentration of 107 cells/mL were used.
Samples were diluted 40-80 fold and measured fluorometrically.
ab65336 Triglyceride Quantification Assay kit (Colorimetric/Fluorometric)
18
RESOURCES
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 TG standard dilution for your desired detection method:
colorimetric [2 – 10 nmol/well] or fluorometric [0.2 – 1 nmol/well].

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 minutes
at RT.

Prepare a master mix for Triglyceride Reaction Mix:
Component
Colorimetric
Reaction Mix (µL)
Fluorometric
Reaction Mix (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 minutes

Measure plate at OD 570 nm for colorimetric assay or at
Ex/Em = 535/587 nm for fluorometric assay.
ab65336 Triglyceride Quantification Assay kit (Colorimetric/Fluorometric)
19
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
Use provided protocol for
deproteinization
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
Improperly thawed
components
Allowing reagents to
sit for extended times
on ice
Incorrect incubation
times or temperatures
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
Thaw all components completely
and mix gently before use
Always thaw and prepare fresh
reaction mix before use
Verify correct incubation times
and temperatures in protocol
ab65336 Triglyceride Quantification Assay kit (Colorimetric/Fluorometric)
20
RESOURCES
Problem
Cause
Solution
Standard
readings do
not follow a
linear pattern
Pipetting errors in
standard or reaction
mix
Air bubbles formed in
well
Avoid pipetting small volumes
(< 5 µL) and prepare a master mix
whenever possible
Pipette gently against the wall of
the tubes
Standard stock is at
incorrect
concentration
Measured at incorrect
wavelength
Always refer to dilutions described
in the protocol
Samples contain
interfering
substances
Sample readings
above/ below the
linear range
Troubleshoot if it interferes with
the kit
Unanticipated
results
Check equipment and filter setting
Concentrate/ Dilute sample so it is
within the linear range
ab65336 Triglyceride Quantification Assay kit (Colorimetric/Fluorometric)
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RESOURCES
17. 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 if the color of the reaction
in the well changes due to the amount used, then it can potentially
interfere with the assay.
18. FAQs
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 570 nm 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.
ab65336 Triglyceride Quantification Assay kit (Colorimetric/Fluorometric)
22
RESOURCES
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.
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.
Can I use Tween 20 or Triton X-100 instead of NP-40 to prepare the
samples?
NP-40 works better to keep lipids in solution and does not create
background for the assay.
ab65336 Triglyceride Quantification Assay kit (Colorimetric/Fluorometric)
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RESOURCES
19. NOTES
ab65336 Triglyceride Quantification Assay kit (Colorimetric/Fluorometric)
24
RESOURCES
ab65336 Triglyceride Quantification Assay kit (Colorimetric/Fluorometric)
25
UK, EU and ROW
Email: [email protected] | Tel: +44-(0)1223-696000
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Tel (Deutsch): 0435-016-424 | Tel (Français): 0615-000-530
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