ab136946 –
Pin1 Human
ELISA Kit
Instructions for Use
For quantitative detection of Peptidyl-prolyl cis-trans isomerase (Pin1)
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. PLATE PREPARATION
9
10
12
13
ASSAY PROCEDURE
13. ASSAY PROCEDURE
14
DATA ANALYSIS
14. CALCULATIONS
15. TYPICAL DATA
16. TYPICAL SAMPLE VALUES
17. ASSAY SPECIFICITY
16
17
18
20
RESOURCES
18.
19.
TROUBLESHOOTING
NOTES
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1
INTRODUCTION
1. BACKGROUND
Abcam’s Pin1 ELISA (Enzyme-Linked Immunosorbent Assay) kit is an
in vitro enzyme-linked immunosorbent assay for the quantitative
measurement of Pin1 in Human cell lysates.
A mouse monoclonal antibody has been precoated onto 96-well plates.
Standards or test samples are added to the wells, and incubated at
room temperature. The wells are washed and a polyclonal detector
antibody specific to Pin1 is added, followed by incubation at room
temperature. After further washing, a horseradish peroxidase (HRP)
conjugated anti-species antibody is added to each well and incubated
at room temperature. After incubation the excess reagents are washed
away. TMB substrate is added to each well and after a short incubation
the enzyme reaction is stopped and the yellow color generated is read
at 450 nm. The intensity of the yellow coloration is directly proportional
to the amount of Pin1 captured in the plate.
Pin1 is also known as dodo, PPIase, PPIase Pin1, Rotamase PIN1,
and Peptidyl-prolyl cistrans isomerase NIMA-interacting 1. A 163
amino acid nuclear protein with a calculated molecular weight of 18.2
kDa, Pin1 has been implicated in neurodegenerative disorders such as
Alzheimer’s Disease1 and is over-expressed in many Human
cancers2, 3. Pin1 is involved in many basic cellular processes
including cell-cycle regulation, transcription, differentiation, and
proliferation4. A member of the parvulin family within the peptidylprolyl
cis-trans isomerase (PPIase) group of proteins, Pin1 contains a Cterminal PPIase domain and an N-terminal WW domain. The WW
domain recognizes a specific motif of phosphorylated serine or
threonine residues preceding a proline. Once bound, Pin1 catalyzes
the cis-trans isomerisation of the proline-containing protein. As a
catalyst involved in oncogenesis, Pin1 is considered a potential
therapeutic target in cancer.
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INTRODUCTION
The reagents in this kit are Human specific. Although there is 96%
homology between Human and mouse and 97% homology between
Human and rat, this kit is only recommended for use with Human and
mouse samples and should not be used for rat samples. Rat samples
have been tested and shown to not work in this assay. Other species
have not been tested.
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3
INTRODUCTION
2. ASSAY SUMMARY
Prepare all reagents, samples and
standards as instructed.
Add standard or sample to each well
used. Incubate at room temperature.
Wash and add prepared detection
antibody to each well. Incubate at
room temperature.
Wash and add prepared antibody-HRP
conjugate. Incubate at room
temperature.
Add TMB Substrate to each well.
Incubate at room temperature. Add
Stop Solution to each well. Read
immediately.
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4
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 Pin1 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 4ºC except the standard which
must be stored at -20ºC.
5. MATERIALS SUPPLIED
Amount
Storage
Condition
(Before
Preparation)
Assay Buffer 10 Concentrate
100 mL
4ºC
Human Pin1 Standard
0.15 mL
-20ºC
RIPA Cell Lysis Buffer 2
100 mL
4ºC
Microplate coated with mouse monoclonal
specific antibody to Human Pin1
96 wells
4ºC
20X Wash Buffer Concentrate
100 mL
4ºC
Human anti-Pin1 polyclonal antibody
10 mL
4ºC
Anti-rabbit IgG-HRP Conjugate
10 mL
4ºC
TMB Substrate
10 mL
4ºC
Stop Solution 2
10 mL
4ºC
Item
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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

Protease inhibitor cocktail (PIC)
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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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

If inhibitors other than those recommended are used, the end user
is responsible for assay validation. In some cases, some protease
inhibitor cocktails may cause performance differences

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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8
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 50 mL of the
supplied Wash Buffer Concentrate with 950 mL of distilled
water. This can be stored at room temperature until the kit’s
expiration date, or for 3 months, whichever comes first.
9.2
1X Assay Buffer 10
Prepare the 1X Assay Buffer 10 by diluting 100 mL of the
supplied concentrate with 900 mL of deionized water. This
can be stored at room temperature until the kit’s expiration
or 3 months, whichever is earlier.
9.3
PIC Addition
Immediately prior to use, PIC must be added to the assay
buffer and RIPA Cell Lysis Buffer 2. If using Sigma Protease
Inhibitor Cocktail, add 0.5 μL/mL PIC, or equivalent
concentration according to alternate vendor’s specification
sheet.
Inhibitors must be freshly added to the assay buffer and
RIPA Cell Lysis Buffer 2 to ensure optimal integrity of the
standards and samples. Each inhibitor treated buffer should
incubate for 5-10 minutes at room temperature before it is
used. Buffers treated with inhibitors should be used within 1
hour of preparation.
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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 Pin1 standards
should be used within 1 hour of preparation.
10.1 Allow the Pin1 standard to equilibrate to room temperature.
10.2 Label seven tubes with numbers 1 – 6.
10.3 Add 500 μL of the Assay Buffer to tubes 2–6.
10.4 Prepare a 2,000 pg/mL Standard 1 by adding 50 µL of the
40,000 pg/mL Stock Standard to 950 μL of the Assay Buffer
into tube 1. Mix thoroughly and gently.
10.5 Prepare Standard 2 by transferring 500 μL from Standard 1
to tube 2. Mix thoroughly and gently.
10.6 Prepare Standard 3 by transferring 500 μL from Standard 2
to tube 3. Mix thoroughly and gently.
10.7 Using the table below as a guide, repeat for tubes 4 through
6.
10.8 Standard 7 contains no protein and is the blank control.
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ASSAY PREPARATION
Standard
#
Sample to
Dilute
1
2
3
4
5
6
7
Stock
Standard 1
Standard 2
Standard 3
Standard 4
Standard 5
None
Volume to
Dilute
(µL)
50
500
500
500
500
500
-
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Volume of
Diluent (µL)
950
500
500
500
500
500
500
Starting
Conc.
(pg/mL)
40,000
2,000
1,000
500
250
125
-
Final
Conc.
(pg/mL)
2,000
1,000
500
250
125
62.5
-
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ASSAY PREPARATION
11. SAMPLE COLLECTION AND STORAGE

This assay is suitable for measuring Pin1 in a wide range of cell
lysates. Human and mouse cell lysates are suitable for use in the
assay. Rat cell lysates are not recommended due to poor sample
recoveries obtained. Prior to assay, frozen samples should be
slowly brought to 4ºC and centrifuged, if necessary, to isolate any
residual cell debris

A minimum 1:8 dilution is required for the RIPA Cell Lysis Buffer
2. This is the minimum dilution required to remove matrix
interference of this buffer. Cell lysates containing more than 20
μg/mL of total cellular protein may require a greater dilution with
assay buffer plus inhibitors to eliminate interference and to be
read in the standard curve
Protocol for Cell Lysis:
1.
Harvest cells and centrifuge at 1,400 rpm for 7 minutes at
4ºC. Discard supernatant.
11.2 Resuspend pellet and wash with Hank’s Balanced Salt
Solution.
11.3 Centrifuge at 1,400 rpm for 7 minutes at 4ºC. Discard
supernatant.
11.4 Resuspend pellet with RIPA Cell Lysis Buffer 2 plus
inhibitors. Vortex and incubate on ice for 30 minutes.
11.5 Centrifuge at 16,000 x g for 20 minutes at 4ºC. The
supernatants can be stored at or below -20ºC or used
immediately in the assay.
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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
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
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
Prepare all reagents, working standards, and samples as
directed in the previous sections.
13.2
Add 100 μL of Standards 1 through 6 into the appropriate
wells.
13.3
Add 100 μL of the Samples into the appropriate wells.
13.4
Seal the plate and incubate for 1 hour on a plate shaker at
500 rpm and at room temperature.
13.5
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.
13.6
Add 100 μL of the Pin1 monoclonal detection antibody to
every well.
13.7
Seal the plate and incubate for 1 hour on a plate shaker at
500 rpm and at room temperature.
13.8
Wash as described in step 13.5.
13.9
Add 100 µL of the anti-rabbit IgG – HRP conjugate to all
wells.
13.10 Seal the plate and incubate for 30 minutes on a plate
shaker (~500 rpm) at room temperature.
13.11 Wash as described in step 13.5.
13.12 Add 100 μL TMB substrate solution to each well.
13.13 Seal the plate and incubate for 30 minutes on a plate
shaker at 500 rpm and at room temperature.
13.14 Add 100 μL Stop Solution into each well.
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ASSAY PROCEDURE
13.15 Read the O.D. absorbance at 450 nm, preferably with
correction between 570 and 590 nm.
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DATA ANALYSIS
14. 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.

Calculate the average net Optical Density (OD) bound for each
standard and sample by subtracting the average blank control OD
from the average OD bound:
Average Net OD = Average Bound OD - Average blank control OD

Plot the average Net OD for each standard versus Human Pin1
concentration in each standard. Sample concentrations may be
calculated off of Net OD values using the desired curve fitting
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DATA ANALYSIS
15. TYPICAL DATA
Data provided for demonstration purposes only. A new standard
curve must be generated for each assay performed.
Sample
Net OD
Standard 1
2.131
Pin1
(pg/mL)
2,000
Standard 2
1.237
1,000
Standard 3
0.72
500
Standard 4
0.483
250
Standard 5
0.345
125
Standard 6
0.273
62.5
Standard 8
0.205
0
Unknown 1
0.959
722.4
Unknown 2
0.303
88.9
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DATA ANALYSIS
16. TYPICAL SAMPLE VALUES
SENSITIVITY –
The sensitivity of the assay, defined as the concentration of Pin1
measured at 2 standard deviations from the mean of 20 zeros along
the standard curve, was determined to be 15.9 pg/mL
LINEARITY OF DILUTION –
A buffer sample containing Pin1 was serially diluted 1:2 in the assay
buffer and measured in the assay. The results are shown in the table
below.
Neat
Expected
(pg/mL)
-
Observed
(pg/mL)
1469.4
Recovery
(%)
-
1:2
750
749.3
99.9
1:4
375
355.1
94.7
1:8
187.5
185.4
98.9
1:16
93.8
85.1
90.8
Dilution
RECOVERY –
Pin1 concentrations were measured in modified RIPA Cell Lysis Buffer
2 and in cell lysates. Pin1 was spiked into the undiluted modified RIPA
Cell Lysis Buffer 2. The cell lysates were run unspiked. These samples
were then diluted with Assay Buffer 10 plus inhibitors and assayed in
the kit.
Recovery
(%)
Recommended
Dilution
107.7
≥1:8
97.70
≥1:275
0.311 mg/mL of Jurkat Lysate
102.00
≥1:21
3.0 mg/mL of mouse 3T3 Lysate
87.70%
≥1:192
Sample
RIPA Cell Lysis Buffer 2
spiked with Pin1 antigen
4.13 mg/mL of MCF-10a Lysate
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DATA ANALYSIS
PRECISION –
Intra-assay precision was determined by assaying 20 replicates of
three buffer controls containing Pin1 in a single assay.
Human Pin1
(pg/mL)
727
366
133
% CV
6.5
3.1
8.1
Inter-assay precision was determined by measuring buffer controls of
varying Pin1 concentrations in multiple assays over several days.
Human Pin1
(pg/mL)
719
403
109
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% CV
5.2
7.7
12.9
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DATA ANALYSIS
17. ASSAY SPECIFICITY
CROSS REACTIVITY –
The cross reactivities for a number of related compounds were
determined by diluting cross reactants in the assay buffer at a
concentration of 200,000 pg/mL. These samples were then measured
in the assay.
Compound
Human Pin1
Β-Catenin
Bcl-2
Bad
PCNA
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Cross Reactivity
(%)
100
<0.02
<0.02
<0.02
<0.02
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RESOURCES
18. 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
19. 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
France
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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RESOURCES
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