ab179842 –
NQO1 Human ELISA Kit
Instructions for Use
For the quantitative measurement of NQO1 in human cell extracts.
This product is for research use only and is not intended for diagnostic
use.
Last Updated 1 July 2015
Table of Contents
INTRODUCTION
1.
BACKGROUND
2.
ASSAY SUMMARY
2
3
GENERAL INFORMATION
3.
4.
5.
6.
7.
8.
PRECAUTIONS
STORAGE AND STABILITY
MATERIALS SUPPLIED
MATERIALS REQUIRED, NOT SUPPLIED
LIMITATIONS
TECHNICAL HINTS
4
4
4
5
5
6
ASSAY PREPARATION
9.
10.
11.
12.
REAGENT PREPARATION
STANDARD PREPARATION
SAMPLE PREPARATION
PLATE PREPARATION
7
8
10
12
ASSAY PROCEDURE
13.
ASSAY PROCEDURE
13
DATA ANALYSIS
14.
15.
16.
17.
18.
CALCULATIONS
TYPICAL DATA
TYPICAL SAMPLE VALUES
SPECIES REACTIVITY
ASSAY SPECIFICITY
15
16
17
18
19
RESOURCES
19.
20.
TROUBLESHOOTING
NOTES
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20
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1
INTRODUCTION
1.
BACKGROUND
Abcam’s NQO1 Human in vitro ELISA (Enzyme-Linked
Immunosorbent Assay) kit is designed for the accurate quantitative
measurement of NQO1 protein in human cell and tissue extracts.
This assay employs an antibody specific for anti- NQO1 coated on the
well a 96- well microplate. Standards and samples are pipetted into the
wells and NQO1 present in a sample is bound to the wells by the
immobilized antibody. The wells are washed to remove any unbound
capture antibody and samples and a NQO1 detector antibody is
pipetted to the wells. After further washing an HRP label is pipetted to
the wells and is incubated. The wells are again washed, a TMB
development solution is added to the wells and color develops in
proportion to the amount of NQO1 bound. Alternatively, a stop solution
which changes the color from blue to yellow, and the intensity of the
color is measured at 450 nm, can be added to the wells.
NQO1 or NAD(P)H dehydrogenase [quinone] 1 is a 31kDa cytoplasmic
enzyme that apparently serves as a quinone reductase in connection
with conjugation reactions of hydroquinones involved in detoxification
pathways as well as in biosynthetic processes such as the vitamin Kdependent gamma-carboxylation of glutamate residues in prothrombin
synthesis. This FAD-binding protein forms homodimers and reduces
quinones to hydroquinones. This protein's enzymatic activity prevents
the one electron reduction of quinones that results in the production of
radical species. Altered expression of this protein has been seen in
many tumors and is also associated with Alzheimer's disease (AD).
Alternate transcriptional splice variants, encoding different isoforms,
have been characterized.
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2
INTRODUCTION
2. ASSAY SUMMARY
Remove appropriate number of
antibody coated well strips. Equilibrate
all reagents to room temperature.
Prepare all the reagents, samples, and
standards as instructed.
Add standard or sample to each well
used. Incubate at room temperature.
Aspirate and wash each well. Add
prepared Detector Antibody to each
well. Incubate at room temperature.
Aspirate and wash each well. Add
prepared HRP label. Incubate at room
temperature.
Aspirate and wash each well. Add
TMB Development Solution to each
well. Immediately begin recording the
color development. Alternatively add a
Stop solution at a user-defined time.
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3
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. Modifications to the kit components or
procedures may result in loss of performance.
4. STORAGE AND STABILITY
Store kit at 4ºC immediately upon receipt.
Refer to list of materials supplied for storage conditions of individual
components. Observe the storage conditions for individual prepared
components in sections 9 & 10.
5. MATERIALS SUPPLIED
Item
Amount
20X Wash Buffer
20 mL
Extraction Buffer
15 mL
10X Blocking Buffer
NQO1 Microplate (12 x 8 well strips)
6 mL
96 wells
NQO1 Human Lyophilized Recombinant Protein
1 µg
10X NQO1 Detector Antibody
1 mL
10X HRP Label
1 mL
HRP Development Solution
12 mL
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Storage
Condition
(Before
Preparation)
4ºC
4ºC
4ºC
4ºC
4ºC
4ºC
4ºC
4ºC
4
GENERAL INFORMATION
6. 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 600 or
450 nm.

Method for determining protein concentration (BCA assay
recommended).

Deionized water.

Multi and single channel pipettes.

PBS (1.4 mM KH2PO4, 8 mM Na2HPO4, 140 mM NaCl, 2.7 mM
KCl, pH 7.3).

Tubes for standard dilution.

Stop solution (optional) – 1N Hydrochloric acid (HCl).

Plate shaker for all incubation steps (optional).

Phenylmethylsulfonyl Fluoride (PMSF)
inhibitors) and/or phosphatase inhibitors.
(or
other
protease
7. 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.
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5
GENERAL INFORMATION
8. TECHNICAL HINTS

Samples generating values higher than the highest standard
should be further diluted in the appropriate sample dilution buffers.

Avoid foaming
components.

Avoid cross contamination of samples or reagents by changing tips
between sample, standard and reagent additions.

Ensure plates are properly sealed or covered during incubation
steps.

Complete removal of all solutions and buffers during wash steps is
necessary to minimize background.

As a guide, typical ranges of sample concentration for commonly
used sample types are shown below in Sample Preparation
(section 11).

All samples should be mixed thoroughly and gently.

Avoid multiply freeze/thaw of samples.

Incubate ELISA plates on a plate shaker during all incubation
steps (optional).

When generating positive control samples, it is advisable to
change pipette tips after each step.

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.
or
bubbles
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when
mixing
or
reconstituting
6
ASSAY PREPARATION
9. REAGENT PREPARATION
Equilibrate all reagents to room temperature (18-25°C) prior to use.
9.1
1X Wash Buffer
Prepare 1X Wash Buffer by adding 20 mL 20X Wash Buffer
to 380 mL nanopure water. Mix gently and thoroughly..
9.2
1X Incubation Buffer
Prepare 1X Incubation Buffer by adding 6 mL 10X Blocking
Buffer to 54 mL 1X Wash Buffer. Mix gently and thoroughly.
9.3
1X NQO1 Detector Antibody
Prepare 1X NQO1 Detector Antibody by diluting the 10X
NQO1 Detector Antibody 10-fold with Incubation Buffer
immediately prior to use. Prepare 500 µL 1X NQO1 Detector
Antibody for each 8 well strip used
9.4
1X HRP Label
Prepare 1X HRP Label by diluting the 10X HRP Label
10-fold with 1X Incubation Buffer immediately prior to use.
Prepare 500 µL 1X HRP Label for each 8 well strip used.
● After opening, the unused Incubation Buffer should be stored at
-20°C.
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7
ASSAY PREPARATION
10. STANDARD PREPARATION
Prepare serially diluted standards immediately prior to use. Always
prepare a fresh set of positive controls for every use.
10.1 Reconstitute the NQO1 human lyophilized recombinant
protein by adding 100 µL deionized water. Allow to sit for 1
minute. This 10X Stock Standard Solution (100 ng/mL)
should be aliquoted in 10 µL aliquots and stored at -80 ºC
10.2 Prepare a 1X Stock Standard Solution (1,000 ng/mL) by
adding 90 µL 1X Incubation Buffer to 10 µL of 10X Stock
Standard Protein Stock.
10.3 Label eight tubes with numbers 1 – 8.
10.4 Add 150 μL Incubation Buffer into tube numbers 2 - 8.
10.5 Prepare a 100 ng/mL Standard #1 by adding 30 µL 1X
Stock Standard Solution to 270 µL 1X Incubation Buffer to
tube #1. Mix thoroughly and gently.
10.6 Prepare Standard #2 by transferring 150 μL Standard #1 to
tube #2. Mix thoroughly and gently.
10.7 Prepare Standard #3 by transferring 150 μL Standard #2 to
tube #2. Mix thoroughly and gently.
10.8 Using the table below as a guide, repeat for tubes #4
through #7.
10.9 Use the diluent as the zero standard tube labeled #8.
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8
ASSAY PREPARATION
Standard
#
Sample to
Dilute
Volume to
Dilute
(µL)
1
2
3
4
5
6
7
1X Stock
Standard #1
Standard #2
Standard #3
Standard #4
Standard #5
Standard #6
30
150
150
150
150
150
150
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Volume
of
Diluent
(µL)
270
150
150
150
150
150
150
Starting
Conc.
(ng/mL)
Final
Conc.
(ng/mL)
1,000
100
50
25
12.5
6.25
3.13
100
50
25
12.5
6.25
3.13
1.56
9
ASSAY PREPARATION
11. SAMPLE PREPARATION
TYPICAL SAMPLE DYNAMIC RANGE –
Typical working ranges
Sample Type
Range (µg/mL)
HepG2
0.5 – 125
MCF7
2.0 – 500
HeLa
2.0 – 500
11.1 Preparation of extracts from cell pellets
11.1.1 Collect non adherent cells by centrifugation or
scrape to collect adherent cells from the culture
flask. Typical centrifugation conditions for cells are
500 x g for 5 minutes at 4ºC.
11.1.2 Rinse cells twice with PBS.
11.1.3 Solubilize cell pellet at 2x107/mL in Extraction Buffer
11.1.4 Incubate on ice for 20 minutes. Centrifuge at
18,000 x g for 20 minutes at 4°C. Transfer the
supernatants into clean tubes and discard the
pellets. Assay samples immediately or aliquot and
store at -80°C. The sample protein concentration in
the extract may be quantified using a protein assay.
11.2 Preparation of extracts from adherent cells by direct
lysis (alternative protocol)
11.2.1 Remove growth media and rinse adherent cells 2
times in PBS.
11.2.2 Solubilize the cells by addition of Extraction Buffer
directly to the plate (use 750 µL - 1.5 mL Extraction
Buffer per confluent 15 cm diameter plate).
11.2.3 Scrape the cells into a test tube and incubate the
lysate on ice for 15 minutes. Centrifuge at
18,000 x g for 20 minutes at 4°C. Transfer the
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10
ASSAY PREPARATION
supernatants into clean tubes and discard the
pellets. Assay samples immediately or aliquot and
store at -80°C. The sample protein concentration in
the extract may be quantified using a protein assay.
11.3 Preparation of extracts from tissue homogenates
11.3.1 Tissue lysates are typically prepared by
homogenization of tissue that is first minced and
thoroughly rinsed in PBS to remove blood (dounce
homogenizer recommended).
11.3.2 Homogenize 100 to 200 mg of wet tissue in
500 µL - 1 mL of the supplied extraction buffer. For
lower amounts of tissue adjust volumes accordingly.
11.3.3 Incubate on ice for 20 minutes. Centrifuge at
18,000 x g for 20 minutes at 4°C. Transfer the
supernatants into clean tubes and discard the
pellets. Assay samples immediately or aliquot and
store at -80°C. The sample protein concentration in
the extract may be quantified using a protein assay.
Note: If using serum-containing sample, fetal bovine serum must be
diluted to <0.4% before the sample can be compatible with this assay.
Alternatively serum can be added to the incubation buffer to the same
concentration as experimental samples as a diluent for the standard
protein. This would act as a control when using serum-containing
samples.
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11
ASSAY PREPARATION
12. PLATE PREPARATION

The 96 well plate strips included with this kit are supplied ready to
use. It is not necessary to rinse the plate prior to adding reagents.

Unused well strips should be returned to the plate packet and
stored at 4°C.

For each assay performed, a minimum of 2 wells must be used as
the zero control.

For statistical reasons, we recommend each sample should be
assayed with a minimum of two replicates (duplicates).

Well effects have not been observed with this assay.
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ASSAY PROCEDURE
13. ASSAY PROCEDURE

Equilibrate all materials and prepared reagents to room
temperature prior to use.

It is recommended to assay all standards, controls and
samples in duplicate.
13.1 Prepare all reagents, working standards, and samples as
directed in the previous sections.
13.2 The samples should be diluted within the working range of
the assay in 1X Incubation Buffer. As a guide, typical
ranges of sample concentration are shown above (section
11). Add 50 µL of each sample. Include a 1X incubation
buffer well as a background control.
13.3 Cover/seal the plate and incubate shaking at 400rpm for two
hours at room temperature.
13.4 Wash each well with 3 x 300 µL 1X Wash Buffer. Wash by
aspirating or decanting from wells then dispensing 300 µL
1X Wash Buffer into each well. Complete removal of liquid
at each step is essential for good performance. After the last
wash invert the plate and blot it against clean paper towels
to remove excess liquid.
13.5 Add 50 µL of 1X NQO1 Detector antibody (step 9.3) to each
well used. Cover/seal the plate and incubate shaking at 400
rpm for one hour at room temperature.
13.6 Wash each well three times.
13.7 Add 50 µL of 1X HRP labeled Secondary Detector antibody
(step 9.4) to each well used. Cover/seal the plate and
incubate shaking at 400 rpm for one hour at room
temperature.
13.8 Repeat Step 13.4
13.9
Add 50 µL TMB Development Solution to each well used
and immediately record the blue color development with
elapsed time in the microplate reader prepared with the
following settings:
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13
ASSAY PROCEDURE
Mode:
Kinetic
Wavelength:
600 nm
Time:
up to 15 min.
Interval:
10 sec. - 1 min.
Shake before and between
Shaking:
readings
Alternative– In place of a kinetic reading, at a user defined time,
record the endpoint OD data at (i) 600 nm or (ii) stop the reaction by
adding 50 µL Stop Solution (see Section 6) to each well and record the
OD at 450 nm.
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14
DATA ANALYSIS
14. CALCULATIONS
Subtract average zero standard reading from all readings. Average the
duplicate readings of the positive control dilutions and plot against their
concentrations. Draw the best smooth curve through these points to
construct a standard curve. Most plate reader software or graphing
software can plot these values and curve fit. A four parameter
algorithm (4PL) usually provides the best fit, though other equations
can be examined to see which provides the most accurate (e.g. linear,
semi-log, log/log, 4 parameter logistic).
Read relative protein
concentrations for unknown samples from the standard curve plotted.
Samples producing signals greater than that of the highest standard
should be further diluted and reanalyzed, then multiplying the
concentration found by the appropriate dilution factor.
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DATA ANALYSIS
15. TYPICAL DATA
TYPICAL STANDARD CURVE – Data provided for demonstration
purposes only. A new standard curve must be generated for each
assay performed.
Standard Curve Measurements
O.D./min (600 nm)
Conc.
(ng/mL)
1
2
3
Mean
O.D./min
0
0.0016
0.0017
0.0017
0.0017
1.56
0.0027
0.0027
0.0024
0.0026
3.13
0.0040
0.0036
0.0036
0.0037
6.25
0.0057
0.0062
0.0060
0.0060
12.5
0.0095
0.0096
0.0090
0.0094
25
0.0183
0.0189
0.0182
0.0185
50
0.0325
0.0332
0.0323
0.0326
100
0.0478
0.0483
0.0477
0.0480
Figure 1. Example of NQO1 standard protein standard curve. The NQO1
standard curve was prepared as described in Section 10. Raw data values are
shown in the table. Background-subtracted data values (mean +/- SD) are
graphed.
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DATA ANALYSIS
16. TYPICAL SAMPLE VALUES
SENSITIVITY The calculated minimal detectable (MDD) dose is 1.28 ng/mL.
RECOVERY –
(Sample spiking in representative sample matrices)
Sample Type
10% FBS Medium
50% Extraction Buffer
Average %
Recovery
132.9
101.0
Range (%)
103.4 – 170.7
93.1 – 113.7
LINEARITY OF DILUTION –
Dilution
No Dilution
1:0.5
1:2
1:4
Interpolated
NQO1 (ng/mL)
34.8
72.4
17.8
8.8
ΔO.D./min read
at 600 nm
24.0
40.5
13.6
7.4
% Expected
Value
100
104.1
102.3
100.9
PRECISION –
n=
%CV
IntraAssay
3
3.7
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InterAssay
3
6.3
17
DATA ANALYSIS
17. SPECIES REACTIVITY
This kit detects NQO1 protein in Human samples only.
Figure 2. Human, mouse, and rat cell lysates (shown respectively left to right on
graph) used at a concentration of 125 µg/mL were analyzed using this kit. Neither
mouse NIH-3T3 nor rat H4IIE cells showed significant response. Backgroundsubtracted data values (mean +/- SD) are graphed. The data shown was
interpolated into the standard curve to show the concentration of NQO1 protein in
each sample.
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DATA ANALYSIS
18. ASSAY SPECIFICITY
Figure 3. Human cell lysates used at a concentration of 62.5 µg/mL were
analyzed using this kit. In agreement with Western blotting data, neither HL-60
nor Hek293 cells showed significant response. The data shown was
interpolated into the standard curve to show the concentration of NQO1 protein
in each sample.
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RESOURCES
19. TROUBLESHOOTING
Problem
Poor standard
curve
Cause
Solution
Inaccurate Pipetting
Check pipettes
Improper standard
dilution
Prior to opening, briefly spin
the stock standard tube and
dissolve the powder
thoroughly by gentle mixing
Incubation times too
brief
Ensure sufficient incubation
times; change to overnight
standard/sample incubation
Inadequate reagent
volumes or improper
dilution
Check pipettes and ensure
correct preparation
Plate is insufficiently
washed
Review manual for proper
wash technique. If using a
plate washer, check all ports
for obstructions
Contaminated wash
buffer
Make fresh wash buffer
Improper storage of
the ELISA kit
Store your reconstituted
standards at -80°C, all other
assay components 4°C.
Keep substrate solution
protected from light
Low Signal
Large CV
Low sensitivity
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RESOURCES
20. NOTES
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UK, EU and ROW
Email: technical@abcam.com | Tel: +44-(0)1223-696000
Austria
Email: wissenschaftlicherdienst@abcam.com | Tel: 019-288-259
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Email: supportscientifique@abcam.com | Tel: 01-46-94-62-96
Germany
Email: wissenschaftlicherdienst@abcam.com | Tel: 030-896-779-154
Spain
Email: soportecientifico@abcam.com | Tel: 911-146-554
Switzerland
Email: technical@abcam.com
Tel (Deutsch): 0435-016-424 | Tel (Français): 0615-000-530
US and Latin America
Email: us.technical@abcam.com | Tel: 888-77-ABCAM (22226)
Canada
Email: ca.technical@abcam.com | Tel: 877-749-8807
China and Asia Pacific
Email: hk.technical@abcam.com | Tel: 108008523689 (中國聯通)
Japan
Email: technical@abcam.co.jp | Tel: +81-(0)3-6231-0940
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All information / detail is correct at time of going to print.
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