ab168538 –
mTOR (pSer2448) ELISA
Kit
Instructions for Use
For the quantitative measurement of phosphorylated Ser2448 of
mTOR protein in cell and tissue lysates.
This product is for research use only and is not intended for diagnostic
use.
Version 1 Last Updated 25/04/2013
Table of Contents
INTRODUCTION
1.
BACKGROUND
2.
ASSAY SUMMARY
2
3
GENERAL INFORMATION
3.
PRECAUTIONS
4.
STORAGE AND STABILITY
5.
MATERIALS SUPPLIED
6.
MATERIALS REQUIRED, NOT SUPPLIED
7.
LIMITATIONS
8.
TECHNICAL HINTS
4
4
4
5
5
6
ASSAY PREPARATION
9.
REAGENT PREPARATION
10. POSITIVE CONTROL PREPARATION
11. SAMPLE PREPARATION
12. PLATE PREPARATION
7
8
9
11
ASSAY PROCEDURE
13. ASSAY PROCEDURE
12
DATA ANALYSIS
14. CALCULATIONS
15. TYPICAL DATA
16. TYPICAL SAMPLE VALUES
17. SPECIES REACTIVITY
18. ASSAY SPECIFICITY
13
14
15
16
17
RESOURCES
19. TROUBLESHOOTING
20. NOTES
20
21
INTRODUCTION
1.
BACKGROUND
Abcam’s mTOR pSer2448 in vitro ELISA (Enzyme-Linked
Immunosorbent Assay) kit is designed for the accurate quantitative
measurement of Ser2448 of mTOR protein in cell and tissue lysates.
Mammalian target of rapamycin (mTOR) is a serine/threonine protein
kinase part of two distinct signaling complexes, mTORC1 and
mTORC2. These two complexes share four proteins (mTOR, mLST8,
DEPTOR, Tti1/tel2), with only mTORC1 containing Raptor and
PRAS40 and mTORC2 containing Rictor, mSin1 and Protor1/2. The
complex mTORC1 (rapamycin sensitive complex) coordinates inputs
from growth factors, stress, energy status, oxygen and amino acids
levels to control processes such as protein and lipid synthesis and
autophagy. The complex mTORC2 is insensitive to nutrients and
rapamycin, but it responds to insulin signaling. It also controls ion
transport and cell shape by targeting serum/glucocorticoid protein
kinase (SGK1) and protein kinase (PKC-α) respectively.
The canonical regulation of mTORC1 occurs through the TSC/Rheb
pathway which receives signals from AKT, AMPK and IKKβ to activate
the complex. Phosphorylation of mTOR at Ser2448 is carried out
directly by AKT kinase as well as p70S6 kinase acting as a feedback
signal. Phosphorylation at this site is a biomarker for the activation
state of the PI-3 kinase pathway as well as the activation status of
mTOR. Activation of mTOR leads to phosphorylation of PRAS40,
raptor and DEPTOR and the consequential activation of mTORC1.
Deregulated signaling of mTOR has been implicated in diseases such
as cancer, metabolic syndrome, neurodegeneration and aging.
Constitutive activation of PI3K-mTORC1 signaling in cancer cells
inhibits autophagy, deregulates protein synthesis via 4E-BP1/eIF4E
and increases de novo lipid synthesis via SREBP1. Similarly mTOR
signaling is a key factor in the regulation of tissue metabolism in the
normal and nutrient overload state affecting the hypothalamus, adipose
tissue, the liver, skeletal muscle and pancreas.
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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 positive control 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 and incubate for 30 minutes. Add
Stop solution and record end point.
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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
10X Wash Buffer
Extraction Buffer
10X Blocking Buffer
mTOR Microplate
10X mTOR (pSer2448) Detector Antibody
10X HRP Label
TMB Development Solution
Stop Solution
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Amount
Storage
Condition
(Before
Preparation)
40 mL
15 mL
4ºC
4ºC
6 mL
96 wells
700 µL
1 mL
12 mL
12 mL
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.

Plate shaker for all incubation steps (optional).

Phenylmethylsulfonyl Fluoride (PMSF) (or other protease
inhibitors) and/or phosphatase inhibitors (sodium fluoride or a
cocktail containing sodium orthovanadate, sodium molybdate,
sodium tartrate and imidazole).
7. LIMITATIONS

Assay kit intended for research use only. Not for use in diagnostic
procedures.

Do not use kit or components if it has exceeded the expiration date
on the kit labels.

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.
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 40 mL 10X Wash Buffer
to 360 mL nanopure water. Mix gently and thoroughly.
9.2
Supplemented Extraction Buffer
Supplement appropriate volume of extraction buffer with
protease and phosphatase inhibitors immediately prior to
use.
9.3
1X Supplemented Incubation Buffer
Prepare Incubation Buffer by adding 4 mL 10X Blocking
Buffer to 36 mL 1X Wash Buffer. Supplement this buffer
with a phosphatase inhibitor cocktail and/or 10 mM Sodium
fluoride. Mix gently and thoroughly.
9.4
1X Incubation Buffer
Prepare Incubation Buffer by adding 2 mL 10X Blocking
Buffer to 18 mL 1X Wash Buffer. Mix gently and thoroughly.
9.5
1X mTOR pSer2448 Detector Antibody
Prepare 1X mTOR pSer2448 Detector Antibody by diluting
the 10X mTOR pSer2448 Detector Antibody 10-fold with
1X Supplemented Incubation Buffer immediately prior to use.
Prepare 500 µL 1X mTOR pSer2448 Detector Antibody for
each 8 well strip used.
9.6
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 10X Blocking buffer and Incubation
Buffer should be stored at -20°C.
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7
ASSAY PREPARATION
10. POSITIVE CONTROL PREPARATION
Prepare serially diluted positive controls immediately prior to use.
Always prepare a fresh set of positive controls for every use. The
levels of phosphorylated Ser2448 of mTOR are low in many standard
cell lines and require induction with pharmacological treatment (see
Specificity section below). In cell lines where levels of are low, mTOR
Ser2448 may be phosphorylated with calyculin A treatment (e.g. HeLa
cells treated with 50 nM calyculin for 15 minutes or NIH3T3 cells
treated with 50 ng/mL of rPDGF for 30 minutes in serum free media).
Use a positive control sample to prepare the dilution series. The
relative levels of pSer2448 of mTOR in other experimental samples
can be interpolated from within this positive control sample series.
10.1 Prepare a positive control sample lysate or extract as
directed in sections 11. Dilute the positive control sample to
an upper concentration limit of the working range of the
assay in the 1X Supplemented Incubation Buffer used to
dilute other experimental samples = Standard #1.
10.2 Label tubes #2-7: Add 150 μL 1X Supplemented Incubation
Buffer into each tube.
10.3 Add 150 μL Standard #1 to tube #2 and mix thoroughly
= Standard #2.
10.4 Transfer 150 μL from Standard #2 to tube #3, mix thoroughly
= Standard #3.
10.5 Repeat for Tubes #4 through #7.
10.6 Use the diluent as the zero standard tube labeled #8.
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8
ASSAY PREPARATION
11. SAMPLE PREPARATION
TYPICAL SAMPLE DYNAMIC RANGE Typical working ranges
Sample Type
Range
HeLa cells treated with calyculin A
8 – 1000 µg/mL
MCF7 cells treated with Phorbol
12-myristate 13-acetate (PMA)
15 – 1000 µg/mL
NIH3T3 cells treated with PDGF
60 – 1000 µg/mL
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 Supplemented
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 (avoid freeze/thaw cycles). The
sample protein concentration in the extract must 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 Supplemented
Extraction Buffer directly to the plate (use
750 µL - 1.5 mL Extraction Buffer per confluent
15 cm diameter plate).
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9
ASSAY PREPARATION
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
supernatants into clean tubes and discard the
pellets. Assay samples immediately or aliquot and
store at -80°C (avoid freeze/thaw cycles). The
sample protein concentration in the extract must 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 Supplemented 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 (avoid freeze/thaw cycles). The
sample protein concentration in the extract must be
quantified using a protein assay
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10
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 plate 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. Contents of
each well can be recorded on the template sheet included in the
Resources section.
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11
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 and samples as directed in the
previous sections.
13.2 The samples should be diluted within the working range of
the assay in 1X Supplemented Incubation Buffer. As a
guide, typical ranges of sample concentration are shown
above (section 11). Add 50 µL of each sample. Also
include a 1X Supplemented Incubation buffer as a
background control.
13.3 Cover/seal the plate and incubate shaking at 400rpm for 2
hours at room temperature.
13.4 Wash each well three times. For each wash, rinse each
well of the plate with 300 µL of 1X Wash Buffer.
13.5 Add 50 µL of 1X mTOR pSer2448 Primary Detector
Antibody (step 9.5) to each well used. Cover/seal the plate
and incubate shaking at 400 rpm for 1 hour at room
temperature.
13.6 Wash each well three times. For each wash, rinse each well
of the plate with 300 µL of 1X Wash Buffer.
13.7 Add 50 µL 1X HRP Labeled Secondary Detector antibody
in 1X Incubation Buffer (step 9.6) to each well used.
Cover/seal the plate and incubate at 400 rpm for 1 hour at
room temperature.
13.8 Wash each well three times. For each wash, rinse each well
of the plate with 300 µL of 1X Wash Buffer.
13.9 Add 100 µL TMB Development Solution to each empty well
and incubate shaking at 200 rpm for 30 minutes at room
temperature away from the light
13.10 Add 100 µL of Stop solution to each well and read end point
at 450 nm in the microplate reader.
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12
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, asymmetric sigmoidal 5
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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13
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. 450 nm
Conc.
(µg/mL)
1
2
3
Mean O.D.
450 nm
0.0
0.28
0.25
0.29
0.27
15.6
0.42
0.38
0.39
0.40
31.3
0.56
0.56
0.54
0.55
62.5
0.82
0.78
0.73
0.78
125
1.06
1.04
1.05
1.05
250
1.32
1.37
1.29
1.33
500
2.07
1.91
1.87
1.95
1000
3.52
3.48
3.45
3.48
Figure 1. Dynamic Range of the assay. The extract was prepared with HeLa
cells treated with 50 nM of calyculin A for 3 hours as described in section 11.2.
Data was plotted on an XY graph in the log scale and an asymmetric sigmoidal
5 parameter logistic algorithm formula was used for curve fitting.
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14
DATA ANALYSIS
16. TYPICAL SAMPLE VALUES
SENSITIVITY Calculated minimum detectable dose = 8 µg/mL (zero dose n=16 +
2 standard deviations) using HeLa cells treated with 50 nM of calyculin
A for 15 minutes.
RECOVERY –
(Sample spiking in representative sample matrices)
Sample Type
Average %
Recovery
Range %
Recovery
50% Culture Media (8 Dilutions)
10% Serum (8 Dilutions)
50% Extraction Buffer (8 Dilutions)
73
72.4
120.7
66.5 – 82.7
61.8 – 94.2
93.7 – 142.9
LINEARITY OF DILUTION –
Linearity of dilution was determined by comparing a dilution series of
extracts prepared from HeLa cells treated with 50 nM calyculin A for 3
hours to extracts prepared with MCF7 cells treated with 200 µM PMA
for 1 hour (starting protein concentration at 500 µg/mL)
Dilution factor
MCF7 PMA Loading
(µg/mL)
% Expected Value
1:2
1:4
1:8
1:16
1:32
250
125
62.5
31.2
15.6
109%
99%
94%
84%
95%
PRECISION –
n=
%CV
Intra-Assay
Inter-Assay
24
4.3%
3
1.8%
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15
DATA ANALYSIS
17. SPECIES REACTIVITY
This kit detects mTOR pSer2448 in human and mouse samples.
Cross reactivity to mouse samples was determined with the mouse cell
line NIH3T3 treated with 50 ng/mL of PDGF A/B recombinant protein
for 30 minutes after overnight serum starvation. Figure 4 shows
performance of this kit on NIH3T3 PDGF treated against DMSO
control.
Figure 2. The mTOR pSer2448 ELISA cross reacts with mouse samples.
NIH3T3 cells were treated with 50 ng/mL of PDGF recombinant protein after
serum starvation or BSA control for 30 minutes at 37°C. Samples were
prepared according to section 11.2. A protein titration of PDGF treated cells
was loaded in triplicate (left panel) alongside 4 replicates of both BSA and
PDGF treated cells at 500 µg/mL. BSA and PDGF samples were interpolated
from the standard curve and results are shown on the right panel. PDGF
increases the levels of phosphorylation at Ser2448 by 2 fold.
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16
DATA ANALYSIS
18. ASSAY SPECIFICITY
The specificity of the assay to measure mTOR phosphorylated at
Ser2448 was demonstrated with the use of λ protein phosphatase (λ
Ppase) on calyculin A treated HeLa cells. The relative levels of the
phosphorylated Ser2448 in extracts decreased dramatically when
treated with 1/100 dilution of the enzyme (Figure 3). This result
matched well with a parallel Western blot analysis (using the kit’s
detector antibody) of the same λ protein phosphatase treated sample
used on the ELISA format (Figure 4).
HeLa cells treated with 50 nM of calyculin for 15 minutes were lysed
with the kit’s extraction buffer without phosphatase inhibitor
supplements and lysate was divided into two vials: Control and λ
Ppase. The control vial was supplemented with 10mM sodium fluoride
and a cocktail containing sodium orthovanadate, sodium molybdate,
sodium tartrate and imidazole (not provided with the kit) and left on ice.
The λ Ppase aliquot was diluted 1:4 in 50 mM Hepes, 100 mM NaCl, 2
mM DTT, 0.01 % Brij 35 pH 7.5 and 1mM MnCl2 (not provided with the
kit). Lambda phosphatase was added at 1/100 dilution and the vial
was incubated at 34˚C for 45 minutes. At the end of the treatment, all
samples were divided into two further vials, one was diluted in SDS
loading buffer and analyzed by Western blotting whereas the other was
diluted in incubation buffer and analyzed with the kit.
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17
DATA ANALYSIS
Figure 3. The mTOR pSer2448 ELISA specifically measures the
phosphorylated protein. Calyculin treated HeLa extracts were left untreated
(control) or treated with 1:100 dilution of λ Ppase at 34˚C. Samples were
loaded at 500, 125 and 30 µg/mL on the plate and measured following the kit’s
protocol. Treatment of calyculin treated HeLa extracts with λ Ppase decreases
the signal by 12 fold at the highest loading.
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18
DATA ANALYSIS
Figure 4. Western blot using capture anti-mTOR and detector anti-mTOR
pSer2448. The detector antibody used in this kit specifically detects the
phosphorylated mTOR as determined by Western blotting. Samples were
loaded on an 8% polyacrylamide gel as follows: (1) Marker (2) calyculin treated
HeLa lysate, (3) calyculin treated HeLa lysate dephosphorylated with 1:100
dilution of λ Ppase at 34˚C, (4) Hek293T cell lysate. Samples were then
diluted in SDS-PAGE buffer and loaded at 40 µg/well. Membranes were
blocked with 20% Blocking Buffer ab126587 in TBST for 1 hour and incubated
with either the detector antibody mTOR pSer2448 or the capture antibody
against total mTOR in 10% blocking buffer ab126587 in TBST overnight.
Labeling was carried out with secondary antibodies conjugated to HRP.
Treatment with calyculin A in HeLa cells induces phosphorylation at Ser2448
and λ Ppase completely dephosphorylates mTOR.
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19
RESOURCES
19. TROUBLESHOOTING
Problem
Cause
Low Signal
Incubation times
too brief
Inadequate
reagent volumes
or improper
dilution
Active
phosphatases in
the extract
Large CV
Incubation times
with TMB too brief
Plate is
insufficiently
washed
Low sensitivity
Solution
Ensure sufficient
incubation times; change
to overnight
standard/sample
incubation
Check pipettes and
ensure correct preparation
Add protein phosphatase
inhibitors into the
Extraction Buffer and into
the Incubation Buffer used
for the sample and the
detector antibody dilutions
Ensure sufficient
incubation time till blue
color develops prior
addition of Stop solution
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 ELISA kit
Store all assay
components 4°C. Keep
substrate solution
protected from light
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20
RESOURCES
20. NOTES
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RESOURCES
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Tel (Deutsch): 0435-016-424 | Tel (Français): 0615-000-530
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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.
RESOURCES
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