ab138883
Glutamate Assay Kit
(Fluorometric)
Instructions for Use
For quantifying Glutamic acid in various
biological samples
This product is for research use only and is not
intended for diagnostic use.
1
Table of Contents
1.
Introduction
3
2.
Protocol Summary
5
3.
Kit Contents
6
4.
Storage and Handling
6
5.
Additional Materials Required
6
6.
Assay Protocol
7
7.
Data Analysis
11
8.
Troubleshooting
12
2
1. Introduction
Glutamic acid is one of the 20 proteinogenic amino acids. The
carboxylate anions and salts of Glutamic acid are known as
glutamates. Glutamate is an important neurotransmitter which plays
a key role in long-term potentiation and is important for learning and
memory. Glutamic acid is the precursor of GABA but has somewhat
the opposite function; it might play a role in the normal function of the
heart and the prostate. As one of the few nutrients that crosses the
blood-brain barrier, Glutamic acid is used in the treatment of
diseases such as depression, ADD and ADHD, fatigue, alcoholism,
epilepsy, muscular dystrophy, mental retardation, and schizophrenia.
ab138883 provides a quick and sensitive method for the
measurement of Glutamic acid in various biological samples. In the
assay, the coupled enzyme system catalyzes the reaction between
L-Glutamic acid and NADP to produce NADPH, which is specifically
recognized by NADPH sensor and recycled back to NADP. A red
fluorescence product is produced during the reaction. The signal can
be read by either a fluorescence microplate reader at Ex/Em = 530570 nm/590-600 nm (optimal Ex/Em = 540 nm/590 nm) or an
absorbance microplate reader at 576±5 nm. With our Glutamate
Assay Kit (Fluorometric), we have detected as little as 1µM Glutamic
acid in a 100 μl reaction volume. The assay is robust, and can be
readily adapted for a wide variety of applications that require the
measurement of Glutamic acid.
3
Kit Key Features

Sensitive: Detect as low as 1 µM of Glutamic acid in
solution.

Continuous: Easily adapted to automation without a
separation step.

Convenient: Formulated to have minimal hands-on time. No
wash step needed.

Non-Radioactive: No special requirements for waste
treatment.
4
2. Protocol Summary
Summary for One 96-well Plate
Prepare assay reaction mixture
Add Glutamic acid standards or test
samples
Incubate at room temperature for 30 minutes - 2 hours
Monitor the fluorescence increase at
Ex/Em = 540/590 nm
Note: Thaw all the kit components to room temperature before
starting the experiment.
5
3. Kit Contents
Components
Component A: Enzyme Mix
Amount
1 bottle (lyophilized
powder)
Component B: Assay Buffer
1 x 10 ml
Component C: NADP
1 vial
Component D: Glutamic Acid
1 vial
Component E: Dilution buffer
1 bottle 10 ml
4. Storage and Handling
Keep at -20°C. Avoid exposure to light.
5. Additional Materials Required

96 or 384-well microplates: Solid black microplates

Fluorescence microplate reader
6
6. Assay Protocol
Note: This protocol is for one 96 - well plate.
A. Preparation of Stock Solutions:
1. Prepare NADP stock solution (200X) by adding 100 L
of Dilution Buffer (Component E) into the vial of NADP
(Component C).
Note: The unused NADP stock solution should be
divided into single use aliquots and stored at -20 oC.
2. Prepare Glutamic acid stock solution (100mM) by adding
200 L of Dilution Buffer (Component E) into the vial of
Glutamic Acid (Component D).
Note: The unused glutamic acid stock solution should be
divided into single use aliquots and stored at -20 oC
B. Preparation of Assay Reaction Mixture:
1. Add 10 mL of Assay Buffer (Component B) into the
bottle of Enzyme Mixture (Component A).
2. Add 50 µL 200X NADP stock solution into the Enzyme
Mixture bottle and mix them well.
7
Note: This glutamic acid assay mixture is enough for
two 96-well plates. The unused glutamic acid assay
mixture should be divided into single use aliquots and
stored at -20 oC.
C. Prepare
serially
diluted
glutamic
acid
standards
(0 to 1 mM):
1. Add 10 μL of glutamic acid stock solution into 990 L
Dilution Buffer (Component E) to generate 1 mM
glutamic acid standard solution.
Note: Diluted glutamic acid standard solution is unstable.
Use within 4 hours.
2. Take 200 μL of 1 mM glutamic acid standard solution to
perform 1:3 serial dilutions to get 300, 100, 30, 10, 3, 1
and 0 μM serially diluted glutamic acid standards.
3. Add serially diluted glutamic acid standards and glutamic
acid containing test samples into a solid black 96-well
microplate as described in Tables 1 and 2.
8
Table 1. Layout of glutamic acid standards and test samples in a
solid black 96-well microplate
BL
BL
TS
TS
….
….
GLU1
GLU1
….
….
….
….
GLU2
GLU2
GLU3
GLU3
GLU4
GLU4
GLU5
GLU5
GLU6
GLU6
GLU7
GLU7
Note: GLU= Glutamic Acid Standards, BL=Blank Control,
TS=Test Samples.
Table 2. Reagent composition for each well
Glutamic Acid
Standards
Blank Control
Serial Dilutions*:
Dilution Buffer :
50 μL
50 μL
Test Sample
50 μL
*Note: Add the serially diluted glutamic acid standards from 1 μM
to 1 mM into wells from GLU1 to GLU7 in duplicate.
9
D. Run Glutamic Acid assay:
1. Add 50 μL of glutamic acid assay mixture into each
well of glutamic acid standard, blank control, and
test samples to make the total glutamic acid assay
volume of 100 µL/well.
Note: For a 384-well plate, add 25 μL of sample and
25 μL of glutamic acid assay mixture into each well.
2. Incubate the reaction at room temperature for 30
minutes to 2 hours, protected from light.
3. Monitor the fluorescence increase by using a
fluorescence plate reader at Ex/Em = 530-570/590600 nm (optimal Ex/Em = 540/590 nm).
Note: The contents of the plate can also be
transferred to a white clear bottom plate and read by
absorbance microplate reader at the wavelength of
576  5 nm. The absorption detection has lower
sensitivity compared to the fluorescence reading.
10
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 glutamic acid reaction.
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.
Figure 1. Glutamic acid dose response was measured with
ab138883 in a black 96-well plate using a microplate reader. As low
as 1 µM glutamic acid was detected with 1 hour incubation.
11
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
12
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)
Use the 10kDa spin column
(ab93349) or Deproteinizing
sample preparation kit
(ab93299)
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
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
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)
13
Standard
curve is not
linear
Not fully thawed kit
components
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
Wait for components to thaw
completely and gently mix prior
use
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 (technical@abcam.com) or phone (select
“contact us” on www.abcam.com for the phone number for
your region).
14
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www.abcam.cn
Japan
Email: technical@abcam.co.jp
Tel: +81-(0)3-6231-0940
www.abcam.co.jp
15
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