ab87546
PicoProbe Acetyl CoA
Assay Kit
Instructions for Use
For the rapid, sensitive and accurate
measurement of Acetyl CoA levels in various
samples.
This product is for research use only and is not
intended for diagnostic use.
Version 4 Last Updated 05 April 2013
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Table of Contents
1.
Overview
3
2.
Protocol Summary
4
3.
Components and Storage
5
4.
Assay Protocol
7
5.
Data Analysis
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6.
Troubleshooting
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1. Overview
Acetyl CoA is a central molecule of metabolism. It carries acetate,
used in the build-up and breakdown of larger molecules. Acetyl CoA
is key in synthetic pathways leading to sesquiterpenes, precursors to
cholesterol and other sterols, flavenoids and other polyketides,
polyenes and long-chain fatty acids. It is the source of the acetyl
group used in histone acetylation. The acetyl group is also
incorporated into a variety of other molecules such as acetylcholine,
melatonin, heme and TCA cycle intermediates.
Abcam’s PicoProbe Acetyl CoA Assay Kit is a highly sensitive assay
for determining Acetyl CoA level in a variety of biological samples. In
the assay, free CoA is quenched then Acetyl CoA is converted to
CoA. The CoA is reacted to form NADH which interacts with
PicoProbe to generate fluorescence (Ex=535/Em=587 nm). The
assay can detect 10-1000 pmol of Acetyl CoA (with detection limit
~0.4 µM) in a variety of samples.
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2. Protocol Summary
Standard Curve Preparation
Sample Preparation
A
CoA Conversion
Add Reaction Mix
Measure Fluorescence
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3. Components and Storage
A. Kit Components
Item
Quantity
Acetyl CoA Assay Buffer
25 mL
PicoProbe
0.2 mL
Conversion Enzyme
0.1 mL
Acetyl CoA Enzyme Mix
0.5 mL
Acetyl CoA Substrate Mix (Lyophilized)
1 vial
CoA Quencher
1 mL
Quench Remover (Lyophilized)
1 vial
Acetyl CoA Standard
(1 µmol, Lyophilized)
1 vial
* Store kit at -20°C, protect from light. Warm Acetyl CoA Assay
Buffer to room temperature prior to using it. Briefly centrifuge all
small vials prior to opening. Read the entire protocol before
performing the assay.
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PICOPROBE: Shipped in DMSO, ready to use as supplied. Thaw by
warming to room temperature. Mix well, store at -20°C.
ACETYL COA SUBSTRATE MIX: Dissolve Substrate Mix with 220 μl
Assay Buffer. Pipette up and down to completely dissolve. Once
reconstituted, store at -20°C. Use within two months.
ACETYL COA STANDARD: Dissolve Acetyl CoA Standard in 100 μl
dH2O to generate 10 mM (10 nmol/μl) Acetyl CoA Standard solution.
Keep cold while in use. Once reconstituted, store at -20°C.
QUENCH REMOVER: Dissolve Quench Remover in 220 μl dH2O.
Keep on ice while in use, store at -20°C.
B. Additional Materials Required

Microcentrifuge

Pipettes and pipette tips

Fluorescent or colorimetric microplate reader

96 well plate

Orbital shaker
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4. Assay Protocol
1. Standard Curve Preparation:
a. 0-1 nmol Range:
Dilute
the
Acetyl
CoA
Standard
100X
to
0.1
mM
(100 pmol/μl) by taking 10 μl into 990 μl dH2O. Dilute a
further 5X to 0.02 mM by adding 100 μl to 400 μl dH2O. Add
0, 10, 20, 30, 40, 50 μl into a series of wells in a 96-well
plate. Adjust volume to 50 μl/well with dH2O to generate 0,
200, 400, 600, 800, 1000 pmol/well Acetyl CoA standard.
b. 0-100 pmol Range:
Dilute
the
Acetyl
CoA
Standard
100X
to
0.1
mM
(100 pmol/μl) by taking 10 μl into 990 μl dH2O. Dilute an
additional 50X to 2 μM (2 pmol/μl) by taking 10 μl into 490 μl
of dH2O. Mix well. Add 0, 10, 20, 30, 40, 50 μl into a series
of standards wells on a 96-well plate. Adjust volume to
50 μl/well with dH2O to generate 0, 20, 40, 60, 80, 100
pmol/well Acetyl CoA standard.
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2. Sample Preparation:
Preparation of Cell Culture Samples:
Cell Lysis Buffer (1X):

20 mM Tris (pH 7.5)

150 mM NaCl

1 mM EDTA

1 mM EGTA

1% Triton X-100

2.5 mM sodium pyrophosphate

1 mM b-Glycerolphosphate

1 mM Na3VO4

1 µg/ml Leupeptin
Note: We recommend adding 1 mM PMSF before use.
Preparing Cell Lysates
a) Aspirate media. If desired, treat cells with the required agent
before harvesting.
b) To harvest cells under non-denaturing conditions, remove
media and rinse cells once with ice-cold PBS.
c) Remove PBS and add 0.5 ml 1X ice-cold Cell Lysis Buffer
plus 1 mM PMSF to each plate (10 cm2) and incubate the
plate on ice for 5 minutes.
d) Scrape cells off the plate and transfer to microcentrifuge
tubes. Keep on ice.
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e) Sonicate 4 times for 5 seconds each on ice.
f)
Microcentrifuge for 10 minutes at 4°C, and transfer the
supernatant to a new tube. The supernatant is the cell
lysate.
g) If necessary, lysate can be stored at –80°C.
h) Once the lysate is ready, follow the deproteinization protocol
as mentioned below and then proceed with the whole
protocol.
Deproteinization protocol:
Enzymes in samples interfere with the assay. You should
deproteinize your sample using a perchloric acid/KOH protocol
as follows:
a) Tissue samples (20-1000 mg) should be frozen rapidly
(liquid N2 or methanol/dry ice), weighed and pulverized.
b) Add 2 µl 1N perchloric acid/mg per sample. KEEP COLD!
c) Homogenize or sonicate thoroughly. Spin homogenate at
10,000 x g for 5-10 minutes.
d) Neutralize supernatant with 3M KHCO3, adding repeated 1 µl
aliquots/10 µl supernate while vortexing. Add until bubble
evolution ceases (2-5 aliquots). Put on ice for 5 minutes
e) Check pH (using 1 µl) is ~6-8. Spin 2 minutes at 10,000 x g
to pellet KClO4.
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f)
Add 10 µl samples into duplicate wells (Sample and
Background) of a 96-well plate; bring volume to 50 µl with
Assay Buffer.
3. Free CoASH and succ-CoA in samples generate background. In
order to correct for this background, add 10 μl of CoASH
Quencher to each Standard, Sample and background sample to
quench free CoA. Incubate for 5 min at room temp. Then add
2 μl of Quench Remover, mix and incubate 5 min. In addition,
run background control for each sample to correct for succ-CoA
or some other forms by omitting the Conversion Enzyme.
4. CoA Conversion:
Make up 50 μl of reaction mix for each well to be tested
(Standard, Sample and Background):
0.1 nmol
Bkgd
40 μl
41 μl
Substrate Mix
2 μl
2 μl
2 μl
2 μl
Conversion Enzyme
1 μl
---
1 μl
---
Enzyme Mix
5 μl
5 μl
5 μl
5 μl
PicoProbe
2 μl
2 μl
0.2 μl
0.2 μl
Buffer
0-100 pmol
Bkgd
41.8 μl 42.8 μl
Mix well. Incubate at 37°C for 10 minutes
5. Measure fluorescence using Ex/Em = 535/589 nm with a plate
reader.
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5. Data Analysis
Correct background by subtracting the value of the zero Acetyl CoA
Standard from all readings. The background reading can be
significant and must be subtracted from sample readings. Determine
Background values for each sample tested and correct Acetyl CoA
values for this background.
Plot the Standard Curve. Apply the sample readings to the Standard
Curve to get the Acetyl CoA amount in the sample wells.
The Acetyl CoA concentrations in the test samples:
Concentration = Ay / Sv (pmol/μl; or nmol/ml; or μM)
Where:
Ay is the amount of Acetyl CoA (pmol) in your sample from the
Standard Curve.
Sv is the sample volume (μl) added to the sample well.
Acetyl CoA molecular weight: 809.6
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Standard curves were generated following the kit protocol.
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6. Troubleshooting
Problem
Reason
Solution
Assay not
working
Assay buffer at
wrong temperature
Assay buffer must not be chilled
- needs to be at RT
Protocol step missed
Plate read at
incorrect wavelength
Unsuitable microtiter
plate for assay
Unexpected
results
Re-read and follow the protocol
exactly
Ensure you are using
appropriate reader and filter
settings (refer to datasheet)
Fluorescence: Black plates
(clear bottoms);
Luminescence: White plates;
Colorimetry: Clear plates.
If critical, datasheet will indicate
whether to use flat- or U-shaped
wells
Measured at wrong
wavelength
Use appropriate reader and filter
settings described in datasheet
Samples contain
impeding substances
Unsuitable sample
type
Sample readings are
outside linear range
Troubleshoot and also consider
deproteinizing samples
Use recommended samples
types as listed on the datasheet
Concentrate/ dilute samples to
be in linear range
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Samples
with
inconsistent
readings
Unsuitable sample
type
Samples prepared in
the wrong buffer
Samples not
deproteinized (if
indicated on
datasheet)
Cell/ tissue samples
not sufficiently
homogenized
Too many freezethaw cycles
Samples contain
impeding substances
Samples are too old
or incorrectly stored
Lower/
Higher
readings in
samples
and
standards
Not fully thawed kit
components
Out-of-date kit or
incorrectly stored
reagents
Reagents sitting for
extended periods on
ice
Incorrect incubation
time/ temperature
Incorrect amounts
used
Refer to datasheet for details
about incompatible samples
Use the assay buffer provided
(or refer to datasheet for
instructions)
Use the 10kDa spin column
(ab93349)
Increase sonication time/
number of strokes with the
Dounce homogenizer
Aliquot samples to reduce the
number of freeze-thaw cycles
Troubleshoot and also consider
deproteinizing samples
Use freshly made samples and
store at recommended
temperature until use
Wait for components to thaw
completely and gently mix prior
use
Always check expiry date and
store kit components as
recommended on the datasheet
Try to prepare a fresh reaction
mix prior to each use
Refer to datasheet for
recommended incubation time
and/ or temperature
Check pipette is calibrated
correctly (always use smallest
volume pipette that can pipette
entire volume)
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Problem
Reason
Solution
Standard
curve is not
linear
Not fully thawed kit
components
Wait for components to thaw
completely and gently mix prior
use
Pipetting errors when
setting up the
standard curve
Incorrect pipetting
when preparing the
reaction mix
Air bubbles in wells
Concentration of
standard stock
incorrect
Errors in standard
curve calculations
Use of other
reagents than those
provided with the kit
Try not to pipette too small
volumes
Always prepare a master mix
Air bubbles will interfere with
readings; try to avoid producing
air bubbles and always remove
bubbles prior to reading plates
Recheck datasheet for
recommended concentrations of
standard stocks
Refer to datasheet and re-check
the calculations
Use fresh components from the
same kit
For further technical questions please do not hesitate to
contact us by email ([email protected]) or phone (select
“contact us” on www.abcam.com for the phone number for
your region).
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www.abcam.co.jp
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