ab65620
Glycogen Assay Kit
Instructions for Use
For the rapid, sensitive and accurate
measurement of Glycogen in various samples.
This product is for research use only and is not
intended for diagnostic use.
1
Table of Contents
1.
Overview
3
2.
Protocol Summary
3
3.
Components and Storage
4
4.
Assay Protocol
6
5.
Data Analysis
9
6.
Appendix
11
7.
Troubleshooting
14
2
1. Overview
Glycogen is the primary short term energy storage molecule in
animals, synthesized primarily in the liver and muscle. Glycogen is a
branched glucose polymer, in α-1,4 linkage, with branching via α-1,6
linkage. Abnormal ability to utilize glycogen is found in diabetes and
in several genetic glycogen storage diseases.
Abcam’s Glycogen Assay Kit is an easy and accurate assay to
measure glycogen levels in biological samples. In the assay,
glucoamylase hydrolyzes the glycogen to glucose which is then
specifically oxidized to produce a product that reacts with OxiRed
probe to generate color (λmax = 570 nm) and fluorescence
(Ex 535/Em 587). This assay can detect glycogen 0.0004 to
2 mg/ml.
2. Protocol Summary
Sample Preparation
Standard Curve Preparation
Add Hydrolysis Enzyme Mix
AAdd Reaction Mix
Measure Optical Density or Fluorescence
3
3. Components and Storage
A. Kit Components
Item
Quantity
Glycogen Hydrolysis Buffer
25 mL
Glycogen Development Buffer
25 mL
OxiRed Probe
0.2 mL
Glycogen Hydrolysis Enzyme Mix (Lyophilized)
1 vial
Glycogen Development Enzyme Mix (Lyophilized)
1 vial
Glycogen Standard (2.0 mg/ml)
100 µL
* Store kit at -20°C, protect from light and moisture.
Warm the Glycogen Assay Buffer to room temperature before use.
Briefly centrifuge all small vials prior to opening.
OXIRED PROBE: Ready to use as supplied. Warm to >18°C to melt
frozen DMSO before use. Mix well, store at -20°C, protect from light
and moisture. Use within 2 months.
4
HYDROLYSIS ENZYME MIX & DEVELOPMENT ENZYME MIX:
Dissolve with 220 μl Hydrolysis Buffer and Development Buffer,
respectively. Vortex tubes gently to dissolve. Keep on ice.
Store at -20°C. Reagents are stable for at least two months.
B. Additional Materials Required

Microcentrifuge

Pipettes and pipette tips

Fluorescent or colorimetric microplate reader

96 well plate

Orbital shaker
5
4. Assay Protocol
1. Sample Preparation:
a. Liquid samples can be assayed directly.
b. For tissue or cell samples: Homogenize 106 cells or 10 mg
tissue with 200 μl dH₂O on ice. Boil the homogenates for 5 min to
inactivate enzymes. Spin the boiled samples at 13000 rpm for 5
min to remove insoluble material; the supernatant is ready for
assay.
Add up to 50 μl of sample or buffer (blank) to test wells. Adjust the
volume to 50 μl with Hydrolysis Buffer.
For unknown samples, we suggest testing several doses of your
sample to ensure the readings are within the standard curve.
Notes:
a) Glycogen can be metabolized very rapidly in some tissues after
death (within a minute), therefore special care must be taken to
minimize glycogen loss when taking tissue samples, such as
freezing samples immediately and keeping cold while working.
6
b) There are a variety of methods for extraction of glycogen from
tissues depending upon the type of tissue or type of information
desired. We strongly recommend consulting the literature to
determine the best method for your purposes. However, for
convenience a few methods taken from literature are described in
section 6.
2. Standard Curve Preparation:
a. For the colorimetric assay:
Dilute the Glycogen Standard to 0.2 mg/ml by adding 10 μl of the
Standard to 90 μl of distilled water, mix well. Add 0, 2, 4, 6, 8,
10 μl to a series of wells. Adjust volume to 50 μl/well with
Hydrolysis Buffer to generate 0, 0.4, 0.8, 1.2, 1.6 and 2.0 μg per
well of the Glycogen Standard.
b. For the fluorometric assay:
Dilute the Glycogen Standard to 0.02 mg/ml by adding 10 μl of
the Standard to 990 μl of distilled water, mix well. Add 0, 2, 4, 6,
8, 10 μl to a series of wells. Adjust volume to 50 μl/well with
Hydrolysis Buffer to generate 0, 0.04, 0.08, 0.12, 0.16 and 0.2 μg
per well of the Glycogen Standard.
3. Hydrolysis:
Hydrolysis Enzyme Mix
Colorimetric
Fluorometric
2 μl
1 μl
7
Add Hydrolysis Enzyme Mix to Standard and samples, mix well,
incubate for 30 min at room temperature.
Note:
Glucose generates background readings. If glucose is present in
your sample, you may do a glucose control without the addition of
hydrolysis enzyme to determine the level of glucose background in
your sample. The glucose background can then be subtracted from
glycogen readings.
4. Development: Mix enough reagents for the number of samples
and standards to be performed: For each well, prepare a total 50 μl
Reaction Mix.
Colorimetric
Fluorometric
Development Buffer
46 μl
48.7 μl
Development Enzyme Mix
2 μl
1.0 μl
OxiRed Probe
2 μl
0.3 μl
Add 50 μl of the Reaction Mix to each well containing Glycogen
Standard or samples.
5: Incubate at room temperature for 30 min, protect from light.
6.
Measure
color
(OD
at
570
nm)
or
fluorescence
(Ex/Em = 535/587 nm).
8
5. Data Analysis
Correct background by subtracting the zero Glycogen control from all
sample readings. The background can be significant and must be
subtracted.
Plot standard curve: μg/well vs. standard readings. Apply sample
readings to the standard curve to get the amount of glycogen in the
sample wells.
The glycogen concentration in the test samples:
Concentration = Ay/Sv (μg/μl, or mg/ml)
Where:
Ay is the amount of glycogen (μg) in your sample from the standard
curve.
Sv is the sample volume (μl) added to the sample well.
Glycogen
(MW
molecular
size:
~60,000
glucose
molecules
~106-107daltons).
Glucose Molecular Weight: 180.16.
9
Glycogen Standard Curve: Assays were performed following the kit
protocol
10
6. Appendix
There are a variety of methods for extraction of glycogen from
tissues depending upon a) the type of tissue the glycogen is to be
extracted form and b) the type of information desired.
A rapid method useful for small tissue samples is as follows:
A small sample of tissue is homogenized in 50 volumes of distilled
water, diluted appropriately and immediately used in the assay.
Since endogenous glucose will be a significant factor utilizing this
method, a glucose background control must be conducted where the
sample is directly placed in development buffer with development
enzyme mix (without prior treatment with the hydrolysis reagents).
If the sample will not be immediately assayed, it should be placed in
a capped, vented microcentrifuge tube and boiled for 5 min to
inactivate any enzyme activities present and stored at -20°C until
assayed.
Samples from high content tissues (liver, muscle) prepared in this
way should have sufficient glycogen such that 5-25 μl aliquots will
give a clearly measureable colorimetric signal. If the sample is from
low content tissues, either take a large aliquot (50μl) for the
colorimetric assay or a proportionally smaller aliquot (10-25μl) in the
fluorometric assay.
11
Caveats:
1) In some tissues such as neural tissue, very rapid rates of
anaerobic metabolism continue after death causing rapid
declines in glucose to undetectable levels within a few seconds.
Utilization of glycogen follows and large decreases in glycogen
content are seen within less than a minute. Thus accurate
measurement of glycogen in such tissues requires very rapid
quenching of metabolic activity such as freeze clamp or
immediate removal of tissue to liquid nitrogen followed by
grinding in the liquid nitrogen and storage at -20 or -80°C until
used.
2) In some samples i.e., Saccharomyces, the glycogen is
distributed between soluble and insoluble pools. It is not clear
that both pools are completely hydrolyzed.
If the sample to be analyzed is sufficiently large (a few hundred
milligrams to grams of tissue), a more quantitative method is as
follows:
Take tissue or cells to a final content of 30-50% in 30% KOH.
Heat to 100°C for 2 hours, cool and add 2 volumes of 95%
ethanol. This will precipitate the crude glycogen. Centrifuge and
collect the precipitate. Dissolve/suspend the precipitate in a
minimal amount of distilled water and acidify to pH 3 with HCL
(5N).
Reprecipitate
with
1
volume
of
ethanol.
Repeat
12
wash/acidification/precipitation
2
more
times,
then
wash
precipitate with ethanol and dry.
This procedure removes the vast majority of the glucose
background with minimal effect on the glycogen. The dried
material can be weighed and dissolved/suspended in hydrolysis
buffer for analysis.
13
7. 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
14
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)
15
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).
16
17
18
UK, EU and ROW
Email: [email protected]
Tel: +44 (0)1223 696000
www.abcam.com
US, Canada and Latin America
Email: [email protected]
Tel: 888-77-ABCAM (22226)
www.abcam.com
China and Asia Pacific
Email: [email protected]
Tel: 108008523689 (中國聯通)
www.abcam.cn
Japan
Email: [email protected]
Tel: +81-(0)3-6231-0940
www.abcam.co.jp
19
Copyright © 2012 Abcam, All Rights Reserved. The Abcam logo is a registered trademark.
All information / detail is correct at time of going to print.