ab176722
Glucose 6 Phosphate
Dehydrogenase Assay
Kit (Fluorometric)
Instructions for Use
For monitoring Glucose 6 Phosphate
Dehydrogenase in a variety of biological
samples.
This product is for research use only and is not
intended for diagnostic use.
Version: 1 Last Updated: 31 October 2013
1
Table of Contents
Table of Contents
2
1.
Overview
3
2.
Protocol Summary
5
3.
Kit Components
6
4.
Storage and Stability
7
5.
Materials Required, Not Supplied
7
6.
Assay Protocol
8
7.
Data Analysis
10
8.
Troubleshooting
12
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1.
Overview
Glucose 6 Phosphate Dehydrogenase (G6PD) catalyzes the
conversion of glucose-6-phosphate to 6-phosphoglucono-δ-lactone,
the first and rate-limiting step in the pentose phosphate pathway. It is
critical metabolic pathway that supplies reducing energy to cells
(such as erythrocytes) by maintaining the level of co-enzyme
nicotinamide adenine dinucleotide phosphate (NADPH), and for the
production of pentose sugars. The production of NADPH is of great
importance for tissues actively engaged in biosynthesis of fatty acids
and/or isoprenoids, such as the liver, mammary glands, adipose
tissue, and the adrenal glands. The NADPH also maintains the level
of glutathione in these cells that helps protect the red blood cells
against oxidative damage. Deficiencies in G6PD predispose
individuals to non-immune hemolytic anemia.
Abcam's
Glucose
6
Phosphate
Dehydrogenase
Assay
Kit
(Fluorometric) (ab176722) provides a simple, sensitive and rapid
fluorescence-based method for detecting G6PD in biological
samples such as serum, plasma, urine, as well as in cell culture
samples. In the enzyme coupled assay, G6PD activity is
proportionally related to the concentration of NADPH that is
specifically monitored by a fluorogenic NADPH sensor to yield a
highly red fluorescence product. The fluorescence signal can be
read with a fluorescence microplate reader at Ex/Em = 540 nm/590
3
nm. With the G6PD assay kit, we were able to detect as little as 0.3
mU/ml G6PD in a 100 µL reaction volume. It is robust, and can be
readily adapted for a wide variety of applications that require the
measurement of G6PD.
2.
Protocol Summary
Prepare G6PD assay mixture
Add G6PD standards or test samples
Incubate at RT for 30 min – 2 hours
Monitor fluorescence intensity at Ex/Em = 540/590 nm
4
3.
Kit Components
Item
Quantity
Storage
upon
arrival
Storage after
use/
reconstitution
Enzyme Probe (lyophilized)
2 bottles
-20°C
-20°C
Assay Buffer
10 mL
-20°C
-20°C
NADP
1 vial
-20°C
-20°C
G6PD Standard 10U
1 vial
-20°C
-20°C
4. Storage and Stability
Upon arrival, store the kit at-20°C and protected from light. Please
read the entire protocol before performing the assay. Avoid repeated
freeze/thaw cycles.
Warm all buffers to room temperature before use. Briefly centrifuge
all small vials prior to opening.
5
5. Materials Required, Not Supplied
 96 or 384-well black plate with clear flat bottoms
 Multi-well spectrophotometer (ELISA reader)
 Distilled water or MilliQ
 PBS
6
6.
Assay Protocol
1. Reagent Preparation
a) Prepare NADP stock solution (100X):
Add 100 μL of H2O into the vial of NADP to make 100X
NADP stock solution. Mix well.
b) Prepare G6PD stock solution:
Add 100 μL of H2O or 1X PBS buffer into the vial of
G6PD Standard (Component D) to make 100 U/mL
G6PD standard solution.
NOTE: The unused G6PD standard stock solution
should be divided into single use aliquots and stored at
-20°C.
2. Prepare serial dilutions of G6PD standard (0 to 300 mU/mL):
a) Add 10 μL of G6PD stock solution into 990 μL 1X PBS buffer
to generate 1000 mU/mL G6PD standard solution.
NOTE: Diluted G6PD standard solution is unstable, and
should be used within 4 hours.
b) Take 200 μL of 1000 mU/mL G6PD standard solution to
perform 1:3 serial dilutions to get 300, 100, 30, 10, 3, 1, 0.3,
and 0 mU/mL serial dilutions of G6PD standard.
c) Add serial dilutions of G6PD standard and G6PD containing
test samples into a solid black 96-well microplate as
described in the tables below.
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BL
G6PD 1
G6PD 2
G6PD 3
G6PD 4
G6PD 5
G6PD 6
G6PD 7
BL
G6PD 1
G6PD 2
G6PD 3
G6PD 4
G6PD 5
G6PD 6
G6PD 7
TS
….
TS
….
….
….
….
….
NOTE: G6PD = Glucose 6 Phosphate Dehydrogenase Standards,
BL = Blank Control, TS = Test Samples.
Reagent composition for each well
Pyrophosphate Standards
Blank Control
Test Sample
Serial Dilutions*: 50 μL
Assay Buffer: 50 μL
50 μL
Note: * Add the serially diluted G6PD standards from 0.3 mU/mL to
300 mU/mL into wells from G6PD1 to G6PD7 in duplicate.
3. Prepare G6PD assay mixture:
a) Add 5 mL of Assay Buffer into one bottle of Enzyme Probe.
b) Add 50 µL NADP stock solution (100X) into the bottle of
Enzyme Probe and mix well.
NOTE: This G6PD assay mixture is enough for one 96-well
plate. The unused assay mixture should be divided into
single use aliquots and stored at -20°C.
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4. Run G6PD assay
a) Add 50 μL of G6PD assay mixture to each well of G6PD
standard, blank control, and test samples to make the total
assay volume of 100 µL/well.
NOTE: For a 384-well plate, add 25 μL of sample and
25 μL assay mixture into each well.
7.
Data Analysis
The fluorescence in blank wells (with the dilution buffer only) is used
as a control, and is subtracted from the values for those wells with
the G6PD reactions. A G6PD standard curve is shown in Figure 1.
Note: The fluorescence background increases with time, thus it is
important to subtract the fluorescence intensity value of the blank
wells for each data point.
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Figure 1. G6PD dose response was measured with Abcam’s
Glucose 6 Phosphate Dehydrogenase Assay Kit (Fluorometric)
(ab176722) in a 96-well black plate using a microplate reader. As
low as 0.3 mU/mL glucose 6 phosphate dehydrogenase in 100 µL
volume can be detected with 1 hour incubation.
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8.
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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Problem
Reason
Solution
Samples
with
inconsistent
readings
Unsuitable sample
type
Refer to datasheet for details
about incompatible samples
Use the assay buffer provided
(or refer to datasheet for
instructions)
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
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
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All information / detail is correct at time of going to print.
Copyright © 2013 Abcam, All Rights Reserved. The Abcam logo is a registered trademark.
All information / detail is correct at time of going to print.