ab136944 – NBR1 ELISA Kit - Abcam · Discover more at 2 INTRODUCTION 1. BACKGROUND Abcam’s NBR1...

Version 1 Last Updated 15 January 2014

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.

ab136944 – NBR1 ELISA Kit

Discover more at www.abcam.com 1

Table of ContentsINTRODUCTION1. BACKGROUND 22. ASSAY SUMMARY 4

GENERAL INFORMATION3. PRECAUTIONS 54. STORAGE AND STABILITY 65. MATERIALS SUPPLIED 66. MATERIALS REQUIRED, NOT SUPPLIED 77. LIMITATIONS 78. TECHNICAL HINTS 8

ASSAY PREPARATION9. REAGENT PREPARATION 910. STANDARD PREPARATIONS 1011. SAMPLE COLLECTION AND STORAGE 1212. SAMPLE PREPARATION 1313. PLATE PREPARATION 14

ASSAY PROCEDURE14. ASSAY PROCEDURE 15

DATA ANALYSIS15. CALCULATIONS 1616. TYPICAL DATA 1717. TYPICAL SAMPLE VALUES 1818. ASSAY SPECIFICITY 20

RESOURCES19. TROUBLESHOOTING 2120. NOTES 22


INTRODUCTION

1. BACKGROUNDAbcam’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 96-well 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


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.


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


GENERAL INFORMATION

3. PRECAUTIONSPlease 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


GENERAL INFORMATION

4. STORAGE AND STABILITYAll components should be kept at -20ºC until the kit’s expiration date. Avoid repeated freeze-thaw cycles.

5. MATERIALS SUPPLIED

Item AmountStorage

Condition(Before

Preparation)Microwell plate coated with anti-NBR1 monoclonal antibody 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


GENERAL INFORMATION

6. MATERIALS REQUIRED, NOT SUPPLIEDThese 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


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


ASSAY PREPARATION

9. REAGENT PREPARATIONEquilibrate all reagents and samples to room temperature (18 - 25°C) prior to use.

9.1 1X Wash BufferPrepare 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.


ASSAY PREPARATION

10.STANDARD PREPARATIONSPrepare 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.


ASSAY PREPARATION

Standard#

Sample toDilute

Volume to Dilute

(µL)

Volume of

Diluent (µL)

StartingConc.

(pg/mL)

Final Conc.

(pg/mL)

1 Standard See Step 10.1 8,0002 Standard 1 300 300 8,000 4,0003 Standard 2 300 300 4,000 2,0004 Standard 3 300 300 2,000 1,0005 Standard 4 300 300 1,000 5006 Standard 5 300 300 500 2507 Standard 6 300 300 250 1258 None - 300 - -


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


ASSAY PREPARATION

12.SAMPLE PREPARATION11 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.


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


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 duplicate13 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.


DATA ANALYSIS

15.CALCULATIONSA 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.


DATA ANALYSIS

16.TYPICAL DATAData provided for demonstration purposes only. A new standard curve must be generated for each assay performed.

Sample NBR1(pg/mL) Net OD

Sample 8 0 0.047

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


DATA ANALYSIS

17.TYPICAL SAMPLE VALUESSENSITIVITY –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.

% Dilutional LinearityDilution Factor HeLa C6 3T3

RIPA2 Lysis Buffer +PIC/PMSF/DNase

1:2 67.5 57.9 62.7 71.31:4 62.5 64.1 67.5 751:8 76.1 85.4 83 95

1:16 88.4 102 106 117

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 DilutionSpike

Concentration (pg/mL)

Recovery of Spike (%)

4,000 89.41,000 87.7

Lysis Buffer + protease inhibitors,

DNase1:8

250 105.9


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.


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 3.71,250 4.4312.5 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 15.61,000 17.2125 21.8

18.ASSAY SPECIFICITYCROSS 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.


RESOURCES

19.TROUBLESHOOTING

Problem Cause Solution

Inaccurate pipetting Check pipettes

Poor standard curve 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 incubationLow Signal

Inadequate reagent volumes or improper

dilution

Check pipettes and ensure correct preparation

Samples give higher value than the highest standard

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 obstructionsLarge CV

Contaminated wash buffer Prepare fresh wash buffer

Low sensitivity

Improper storage of the kit

Store the all components as directed


RESOURCES

20.NOTES

RESOURCES 23

UK, EU and ROWEmail: [email protected] | Tel: +44-(0)1223-696000

AustriaEmail: [email protected] | Tel: 019-288-259

FranceEmail: [email protected] | Tel: 01-46-94-62-96 GermanyEmail: [email protected] | Tel: 030-896-779-154 SpainEmail: [email protected] | Tel: 911-146-554 SwitzerlandEmail: [email protected] Tel (Deutsch): 0435-016-424 | Tel (Français): 0615-000-530

US and Latin AmericaEmail: [email protected] | Tel: 888-77-ABCAM (22226)

CanadaEmail: [email protected] | Tel: 877-749-8807

China and Asia Pacific Email: [email protected] | Tel: 108008523689 (中國聯通) JapanEmail: [email protected] | Tel: +81-(0)3-6231-0940 www.abcam.com | www.abcam.cn | www.abcam.co.jp

Copyright © 2013 Abcam, All Rights Reserved. The Abcam logo is a registered trademark.

All information / detail is correct at time of going to print.

ab136944 – NBR1 ELISA Kit - Abcam · Discover more at 2 INTRODUCTION 1. BACKGROUND Abcam’s NBR1...

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