ab136944 –
NBR1 ELISA Kit
Instructions for Use
For quantitative detection of NBR1 in cell lysates.
This product is for research use only and is not intended for diagnostic
use.
Version 1 Last Updated 15 January 2014
Table of Contents
INTRODUCTION
1.
BACKGROUND
2.
ASSAY SUMMARY
2
4
GENERAL INFORMATION
3.
PRECAUTIONS
4.
STORAGE AND STABILITY
5.
MATERIALS SUPPLIED
6.
MATERIALS REQUIRED, NOT SUPPLIED
7.
LIMITATIONS
8.
TECHNICAL HINTS
5
6
6
7
7
8
ASSAY PREPARATION
9.
REAGENT PREPARATION
10. STANDARD PREPARATIONS
11. SAMPLE COLLECTION AND STORAGE
12. SAMPLE PREPARATION
13. PLATE PREPARATION
9
10
12
13
14
ASSAY PROCEDURE
14. ASSAY PROCEDURE
15
DATA ANALYSIS
15. CALCULATIONS
16. TYPICAL DATA
17. TYPICAL SAMPLE VALUES
18. ASSAY SPECIFICITY
16
17
18
20
RESOURCES
19.
20.
TROUBLESHOOTING
NOTES
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1
INTRODUCTION
1. BACKGROUND
Abcam’s NBR1 ELISA (Enzyme-Linked Immunosorbent Assay) kit is
an in vitro enzyme-linked immunosorbent assay for the quantitative
measurement of NBR1 in Human, mouse and rat NBR1 samples in cell
lysates.
A NBR1 specific monoclonal antibodies have been precoated onto 96well plates. Standards and test samples are added to the wells and the
microplate is then incubated at room temperature. After washing the
wells, an anti- NBR1 antibody conjugated to Horseradish Peroxidase
(HRP) is added. After further incubation, the wells are washed again to
remove unbound antibody conjugate. A TMB substrate solution is then
added after further incubation the enzyme reaction is stopped. The
degree of color change in each well is directly proportional to the
amount of NBR1 captured in that well.
The generic term ‘‘autophagy’’ comprises several processes by which
the lysosome acquires cytosolic cargo, with three types of autophagy
being discerned in the literature:
1. Macroautophagy, characterized by the formation of a
crescent‐shaped structure (the phagophore) that expands to
form the double‐membrane autophagosome, capable of fusion
with the lysosome.
2. Microautophagy, in which lysosomes invaginate and directly
sequester cytosolic components.
3. Chaperone‐mediated autophagy (CMA), which involves
translocation of unfolded proteins across the lysosomal
membrane.
Upregulation of autophagy pathways occurs in response to extra‐ or
intracellular stress and signals such as starvation, growth factor
deprivation, ER stress and pathogen infection. Malfunction of these
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2
INTRODUCTION
pathways is linked to various Human pathologies including cancer,
neurodegeneration and infectious diseases.
Selective macroautophagy describes the pathway of self‐degradation
of whole cellular components, protein aggregates or unusually
long‐lived proteins; in which double membrane autophagosomes
sequester organelles, ubiquitinylated proteins or ubiquitinylated protein
aggregates and subsequently fuse with lysosomes for breakdown by
resident hydrolases. Autophagic clearance of protein aggregates
requires the ubiquitinbinding receptors p62 and NBR1.
NBR1 (neighbor of BRCA1 gene 1), a 966‐amino acid long protein, is a
selective (macro) autophagy substrate that interacts with mono‐ and
poly‐ubiquitin conjugates (K63 and K48‐linked) via its UBA domain,
and LC3/GABARAP via its LIR domain. NBR1 and p62 share very
similar domain organizations; the PB1 (Phox and Bem1) domain of
NBR1 can bind to the PB1 domain of p62, where it either adds to the
polymeric p62 chain or becomes the chain terminus. In spite of the
similarities between the two, p62 and NBR1 do not require each other
for functionality. NBR1 has been detected in Ub‐ and p62‐positive
Mallory bodies in patients with alcoholic steatohepatitus and has been
implicated as a potential biomarker for certain hereditary muscle
diseases and various proteinopathies involving accumulation of
misfolded proteins. NBR1 has also been shown to be a negative
regulator of postnatal osteoblastic bone formation and p38 MAPK
activity.
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3
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 HRP labeled secondary
detector antibody. Incubate at room
temperature
Aspirate and wash each well. Add
TMB Substrate Solution to each well.
Immediately begin recording the color
development
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GENERAL INFORMATION
3. PRECAUTIONS
Please read these instructions carefully prior to beginning the
assay.

Stop Solution 2 is a 1 normal (1N) hydrochloric acid solution. This
solution is caustic; care should be taken in use

The activity of the Horseradish peroxidase conjugate is affected by
nucleophiles such as azide, cyanide and hydroxylamine.

We test this kit’s performance with a variety of samples, however it
is possible that high levels of interfering substances may cause
variation in assay results

The NRB1 standard should be handled with care due to the
unknown effects of the antigen
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GENERAL INFORMATION
4. STORAGE AND STABILITY
All components should be kept at -20ºC until the kit’s expiration
date. Avoid repeated freeze-thaw cycles.
5. MATERIALS SUPPLIED
Amount
Storage
Condition
(Before
Preparation)
96 wells
-20ºC
Assay Buffer 13
50 mL
-20ºC
HRP conjugated monoclonal antibody to NBR1
10 mL
-20ºC
20X Wash Buffer Concentrate
30 mL
-20ºC
NBR1 Standard
2 Vials
-20ºC
TMB Substrate
10 mL
-20ºC
Stop Solution 2
10 mL
-20ºC
RIPA Cell Lysis Buffer 2
100 mL
-20ºC
Item
Microwell plate coated with anti-NBR1
monoclonal antibody
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GENERAL INFORMATION
6. MATERIALS REQUIRED, NOT SUPPLIED
These materials are not included in the kit, but will be required to
successfully utilize this assay:

Standard microplate reader - capable of reading at 450 nm,
preferably with correction between 570 and 590 nm

Automated plate washer (optional)

Adjustable pipettes and pipette tips. Multichannel pipettes are
recommended when large sample sets are being analyzed

Eppendorf tubes

Microplate Shaker

Absorbent paper for blotting

Deionized water

Phenylmethanesulfonyl fluoride (PMSF)

Protease inhibitor cocktail (PIC)

DNase
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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7
GENERAL INFORMATION
8. TECHNICAL HINTS







Standards must be made up in polypropylene tubes
Pre-rinse the pipette tip with the reagent, use fresh pipette tips for
each sample, standard and reagent
Pipette standards and samples to the bottom of the wells
Add the reagents to the side of the well to avoid contamination
This kit uses break-apart microtiter strips, which allow the user to
measure as many samples as desired. Unused wells must be kept
desiccated at 4°C in the sealed bag provided. The wells should be
used in the frame provided
Prior to addition of substrate, ensure that there is no residual wash
buffer in the wells. Any remaining wash buffer may cause variation
in assay results
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
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ASSAY PREPARATION
9. REAGENT PREPARATION
Equilibrate all reagents and samples to room temperature (18 - 25°C)
prior to use.
9.1
1X Wash Buffer
Prepare the 1X wash buffer by diluting 30 mL of the supplied
20X Wash Buffer Concentrate with 570 mL of distilled water.
This can be stored at room temperature until the kit’s
expiration date, or for 3 months, whichever comes first.
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ASSAY PREPARATION
10. STANDARD PREPARATIONS
Prepare serially diluted standards immediately prior to use. Always
prepare a fresh set of standards for every use. Diluted NBR1 standards
should be used within 1 hour of preparation.
10.1 Allow the 8 ng NBR1 standard to equilibrate to room
temperature. Reconstitute one vial of 8 ng NBR1 lyophilized
standard with 1 mL of appropriate diluent (either Assay
Buffer 13 or Tissue Culture Media) to create an 8,000 pg/mL
Standard 1 Solution (see table below).
10.2 Label eight tubes with numbers 2 – 7.
10.3 Add 300 μL appropriate diluent to all other tubes (2–7).
10.4 Prepare a 4,000 pg/mL Standard 2 by transferring 300 µL
from Standard 1 to tube 2. Mix thoroughly and gently.
10.5 Prepare Standard 3 by transferring 300 μL from Standard 2
to tube 3. Mix thoroughly and gently.
10.6 Prepare Standard 4 by transferring 300 μL from Standard 3
to tube 4. Mix thoroughly and gently.
10.7 Using the table below as a guide, repeat for tubes 5 through
7.
10.8 Standard 8 contains no protein and is the Blank Activity
control.
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ASSAY PREPARATION
Standard
#
Sample to
Dilute
1
2
3
4
5
6
7
8
Standard
Standard 1
Standard 2
Standard 3
Standard 4
Standard 5
Standard 6
None
Volume
to Dilute
(µL)
300
300
300
300
300
300
-
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Volume
Starting
of
Conc.
Diluent
(pg/mL)
(µL)
See Step 10.1
300
8,000
300
4,000
300
2,000
300
1,000
300
500
300
250
300
-
Final
Conc.
(pg/mL)
8,000
4,000
2,000
1,000
500
250
125
-
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ASSAY PREPARATION
11. SAMPLE COLLECTION AND STORAGE

This kit is compatible with Human, mouse and rat NBR1 samples
in cell lysates. Samples diluted sufficiently into the assay buffer
can be read directly from a standard curve. Samples containing a
visible precipitate must be clarified prior to use in the assay. Cell
lysates should be diluted at a minimum of 1:8 to avoid lysis buffer
interference in the assay

Samples in a variety of lysis buffers other than that provided in the
NBR1 kit can also be read in the assay provided the standards
have been diluted into the same lysis buffer instead of Assay
Buffer 13 (included in kit). Users should only use standard curves
generated in diluted lysis buffer or assay buffer to calculate
concentrations of NBR1 in the appropriate matrix. It is up to the
end user to validate the use of any lysis buffer other than that
provided in the NBR1 kit

Experimentally observed concentrations of NBR1 protein in cell
lysates may vary due to cell culture/treatment conditions and/or
alterations in lysis procedures. Variations may be caused by, but
are not limited to, one or more of the following: cell type/species,
frequency of media changes, concentration of chemical treatment,
treatment duration, media supplements, and cell confluency.
Interpretation of experimental data should include considerations
of these sources of variability
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ASSAY PREPARATION
12. SAMPLE PREPARATION
11
Centrifuge at 1700 x g for 10 minutes at room temperature
to pellet cells and/or cellular debris.
12.2 Adding protease inhibitors to RIPA Cell Lysis Buffer 2: a.
Add 0.5uL of protease inhibitor cocktail (PIC) per mL of
lysis buffer and add PMSF to a final concentration of 1mM.
Add DNase to a final concentration of 20ug/mL. Inhibitors
must be added fresh just prior to lysis. RIPA 2 Lysis Buffer
containing inhibitors cannot be stored for later use.
12.3 Resuspend cell pellet in lysis buffer with inhibitors and
DNase and incubate on ice for 30 minutes. Vortex
occasionally.
12.4 Pellet cellular debris via centrifugation at 20,000 x g for 10
minutes.
12.5 Divide the lysates into aliquots and store at or below -20°C,
or use immediately in the assay.
12.6 Refer to Sample Handling section for minimum required
dilution (MRD). Avoid repeated freeze-thaw cycles.
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ASSAY PREPARATION
13. 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
blanks, omitting primary antibody from well additions

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. Contents of
each well can be recorded on the template sheet included in the
Resources section
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ASSAY PROCEDURE
14. 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
Prepare all reagents, working standards, and samples as
directed in the previous sections.
14.2
Add 100 μL of each Standard into the appropriate wells.
14.3
Add 100 μL of the Samples into the appropriate wells.
14.4
Seal the plate and incubate for 1 hour on a plate shaker at
500 rpm and at room temperature.
14.5
Empty the contents of the wells and wash by adding 400 µL
of 1X Wash Buffer to every well. Repeat the wash 3 more
times for a total of 4 Washes. After the final wash, empty or
aspirate the wells, and firmly tap the plate on a lint free
paper towel to remove any remaining wash buffer.
14.6
Add 100 μL of the anti-NRB1 monoclonal antibody
conjugated to HRP to every.
14.7
Seal the plate and incubate for 30 minutes on a plate
shaker at 500 rpm and at room temperature.
14.8
Empty and wash the wells as described in step 14.5.
14.9
Add 100 μL TMB substrate solution to each well.
14.10 Seal the plate and incubate for 30 minutes on a plate
shaker at 500 rpm and at room temperature.
14.11 Add 100 μL Stop Solution 2 to each well.
14.12 Read the O.D. absorbance at 450 nm, preferably with
correction between 570 and 590 nm.
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DATA ANALYSIS
15. CALCULATIONS
A four parameter algorithm (4PL) 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). Interpolate 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
16. TYPICAL DATA
Data provided for demonstration purposes only. A new standard
curve must be generated for each assay performed.
Sample 8
NBR1
(pg/mL)
0
Sample 7
125
0.092
Sample 6
250
0.132
Sample 5
500
0.211
Sample 4
1,000
0.379
Sample 3
2,000
0.698
Sample 2
4,000
1.382
Sample 1
8,000
2.587
Sample
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Net OD
0.047
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DATA ANALYSIS
17. TYPICAL SAMPLE VALUES
SENSITIVITY –
The sensitivity or limit of detection of the assay is 65.57 pg/mL. The
sensitivity was determined by interpolation at 2 standard deviations
above the mean signal at background (0 pg/mL) using data from at
least 20 low standards and zeros.
LINEARITY OF DILUTION –
The minimum required dilution for several common samples was
determined by serially diluting samples into the provided lysis buffer
and identifying the dilution at which linearity was observed. As a
control, Tris based buffer with 1% Triton, 0.1% SDS, and 0.5% sodium
deoxycholate (RIPA Lysis buffer 2, catalog number 80‐1284) was
spiked with recombinant NBR1 and diluted in Assay Buffer.
Dilution
Factor
1:2
1:4
1:8
1:16
HeLa
67.5
62.5
76.1
88.4
% Dilutional Linearity
RIPA2 Lysis Buffer
C6
3T3
+PIC/PMSF/DNase
57.9
62.7
71.3
64.1
67.5
75
85.4
83
95
102
106
117
RECOVERY –
After diluting RIPA Cell Lysis Buffer 2 (with Protease Inhibitors and
DNase) to its minimum required dilution, recombinant NBR1 was
spiked at high, medium, and low concentrations and read in the assay.
The recovery of the standard in spiked Lysis Buffer was determined by
interpolation of the resulting net OD values from the standard curve.
Sample Matrix
Minimum
Dilution
Lysis Buffer +
protease inhibitors,
DNase
1:8
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Spike
Concentration
(pg/mL)
4,000
1,000
250
Recovery of
Spike (%)
89.4
87.7
105.9
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DATA ANALYSIS
PARALLELISM –
Parallelism experiments were carried out to determine if the
recombinant NBR1 standard accurately determines NBR1
concentrations in biological matrices. HeLa, 3T3 and C6 cells were
lysed in RIPA Cell Lysis Buffer 2. Values were obtained using the cell
lysates serially diluted in assay buffer and assessed from a standard
curve using four parameter logistic curve fitting. The observed values
were plotted against the dilution factors. Parallelism of the curves
demonstrates that the antigen binding characteristics are similar
enough to allow the accurate determination of native analyte levels in
diluted samples from cell lines of Human, mouse and rat origin.
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DATA ANALYSIS
PRECISION –
Intra‐assay precision was determined by assaying 20 replicates of
three buffer controls containing NBR1 in a single assay.
NBR1
(pg/mL)
% CV
5,000
1,250
312.5
3.7
4.4
7.8
Inter‐assay precision was determined by measuring buffer controls of
varying NBR1 concentrations in multiple assays over several days.
NBR1
(pg/mL)
% CV
8,000
1,000
125
15.6
17.2
21.8
18. ASSAY SPECIFICITY
CROSS REACTIVITY –
The cross reactivity of p62 was determined by diluting it in the assay
buffer at a concentration of 40-400 ng/mL. Samples were then
measured in the assay. No cross reactivity was detected.
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RESOURCES
19. TROUBLESHOOTING
Problem
Poor
standard
curve
Low Signal
Samples
give higher
value than
the highest
standard
Cause
Solution
Inaccurate pipetting
Check pipettes
Improper standards
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
Starting sample
concentration is too
high.
Dilute the specimens and repeat
the assay
Plate is insufficiently
washed
Review manual for proper wash
technique. If using a plate washer,
check all ports for obstructions
Contaminated wash
buffer
Prepare fresh wash buffer
Improper storage of
the kit
Store the all components as
directed
Large CV
Low
sensitivity
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RESOURCES
20. NOTES
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Email: technical@abcam.com
Tel (Deutsch): 0435-016-424 | Tel (Français): 0615-000-530
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Canada
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Japan
Email: technical@abcam.co.jp | Tel: +81-(0)3-6231-0940
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