ab168539 –
mTOR (Total & pSer2448)
In-Cell ELISA Kit (IR)
Instructions for Use
For measuring in high throughput levels of mTOR total protein and
phosphorylated at Ser2448.
This product is for research use only and is not intended for diagnostic
use.
Version 2 Last Updated 19/04/2013
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. SAMPLE PREPARATION
7
9
ASSAY PROCEDURE
11.
ASSAY PROCEDURE
11
DATA ANALYSIS
12. CALCULATIONS
13. TYPICAL DATA
14. TYPICAL SAMPLE VALUES
15. MOUSE REACTIVITY
16. ASSAY SPECIFICITY
13
14
15
15
16
RESOURCES
17. FREQUENTLY ASKED QUESTIONS
18. TROUBLESHOOTING
19. NOTES
19
21
22
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1
INTRODUCTION
1. BACKGROUND
Abcam’s mTOR (Total & pSer2448) In-Cell ELISA (Enzyme-Linked
Immunosorbent Assay) kit is designed to measure levels of mTOR
total protein and phosphorylated at Serine 2448 in cultured cells.
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 and deregulates protein synthesis via 4EBP1/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
Seed cells in a microwell culture plate.
Fix cells with 4% paraformaldehyde for 10 minutes and wash.
Incubate plate with Antigen Retrieval Buffer at 80°C for 15 minutes
and wash
Permeabilize/Block cells for 2 hours at room temperature
Incubate cells with primary antibodies for 2 hours at room
temperature or overnight at 4ºC and wash.
Incubate cells for 2 hours with secondary antibodies and wash
Scan the plate
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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 section 9. Reagent Preparation.
Upon receipt spin down the contents of the Primary Antibody Cocktail
and the IRDye®-Labeled Secondary Antibody tube and protect from
light.
5. MATERIALS SUPPLIED
100 mL
Storage
Condition
(Before
Preparation)
4ºC
400X Tween - 20
2 mL
4ºC
100X Triton X-100
500 µL
4ºC
10X Blocking Buffer
10 mL
4ºC
Antigen Retrieval Buffer
100X mTOR (Total and pSer2448) Primary
Antibody Cocktail Stock
500X IRDye®-Labeled Secondary Antibody
Cocktail (anti-Mouse IRDye800® and anti-Rabbit
IRDye680®)
Janus Green Stain
25 mL
4ºC
120 µL
4ºC
30 µL
4ºC
17 mL
4ºC
Item
10X Phosphate Buffered Saline (PBS)
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Amount
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:

A LI-COR® Odyssey® or Aerius® infrared imaging system.

96 or 384-well amine coated plate(s).

20% paraformaldehyde.

Nanopure water or equivalent.

Water bath

Microplate-adaptable heating block

Multi and single channel pipettes.

0.5 M HCl (optional for Janus Green cell staining procedure).

Optional humid box for overnight incubation step.

Optional plate shaker for all incubation steps.
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 and reagent additions.

Ensure plates are properly sealed or covered during incubation
steps.

Complete removal of all solutions and buffers during wash steps.

During development of this assay we have observed edge effects
after treatment with Antigen Retrieval Buffer. Use perimeter wells
of the plate as control wells (primary antibody omitted).
Regardless, it is required to leave at minimum one well from which
the primary antibodies are excluded to determine background
signals of the assay.
or
bubbles
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when
mixing
or
reconstituting
6
ASSAY PREPARATION
9. REAGENT PREPARATION
Equilibrate all reagents and samples to room temperature (18-25°C)
prior to use.
9.1
1X PBS
Prepare 1X PBS by diluting 60 mL of 10X PBS in 540 mL of
nanopure water or equivalent. Mix well. Store at room
temperature.
9.2
1X Wash Buffer
Prepare 1X Wash Buffer by diluting 750 µL of 400X
Tween-20 in 300 mL of 1X PBS. Mix well. Store at room
temperature.
9.3
8% Paraformaldehyde Solution
Immediately prior to use prepare 8% Paraformaldehyde
Solution in PBS. To make 8% Paraformaldehyde, combine
6 mL of nanopure water or equivalent, 1.2 mL of 10X PBS
and 4.8 mL of 20% Paraformaldehyde.
Note – Paraformaldehyde is toxic and should be prepared
and used in a fume hood. Dispose of paraformaldehyde
according to local regulations.
9.4
Antigen Retrieval Buffer
Pre-heat Antigen Retrieval Buffer to 80⁰C in a water bath
before starting assay. A microplate heating block should also
be pre-heated to 80⁰C.
9.5
1X Permeabilization & Blocking Solution
Immediately prior to use prepare 1X Permeabilization &
Blocking Solution by diluting 500 µL of 100X Triton X-100,
5 mL of 10X Blocking Buffer in 44.5 mL of 1X PBS. Mix well.
9.6
1X Primary Antibody Cocktail Solution
Prepare 1X Primary Antibody Cocktail Solution by diluting
the 100X Primary Antibody Cocktail Stock into appropriate
volume of 1X Permeabilization & Blocking Solution (i.e.
12 mL of solution + 120 µL of the 100X Anti-mTOR( total and
pSer2448) Antibody Cocktail Solution).
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7
ASSAY PREPARATION
Note – The primary antibody cocktail is a mixture of mouse
anti-mTOR total and rabbit anti-mTOR pSer2448 antibodies
9.7
1X Secondary Antibody Cocktail Solution
Prepare 1X Secondary Antibody Cocktail Solution
(anti-Mouse IRDye800® and anti-Rabbit IRDye680®) by
diluting the cocktail stock 500X into an appropriate volume of
1X Permeabilization & Blocking Solution.
Note – This 1X cocktail should be made immediately prior to
adding to microplate.
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8
ASSAY PREPARATION
10. SAMPLE PREPARATION
General Sample information:
● The protocol below is described for a 96-well plate. If performing
assay on a 384-well plate, adjust volumes accordingly.
10.1 Preparation of adherent cells
10.1.1 Seed adherent cells directly into an amine coated
plate and allow them to attach for >6 hours or
overnight. It is advised to seed in a 100 µL volume
of the same media used to maintain the cells in bulk
culture. The optimal cell seeding density is cell type
dependent. The goal is to seed cells such that they
are just reaching confluency (but not over-confluent)
at the time of fixation. As an example, HeLa cells
may be seeded at ~ 100,000 cells per well and
cultured overnight for fixation the following day.
10.1.2 The attached cells can be treated if desired with a
drug of interest.
10.1.3 Fix cells by adding a final concentration of 4%
Paraformaldehyde Solution. This can be achieved
by one of two means: (1) Add an equal volume of
8% Paraformaldehyde Solution to the culture volume
(e.g. add 100 µL 8% Paraformaldehyde to a well
with 100 µL media) or (2) gently remove culture
media from the wells and replace with 100 µL 4%
Paraformaldehyde Solution.
10.1.4 Incubate for 10-20 minutes at room temperature.
10.1.5 Gently remove the Paraformaldehyde Solution from
the plate and wash the plate three times briefly with
1X PBS. For each wash, rinse each well of the plate
with 200 µL of 1X PBS. Finally, add 100 µL of 1X
PBS to the wells of the plate. The plate can now be
stored at 4°C for several days. Cover the plate with
a lid or seal while stored. For prolonged storage
supplement PBS with 0.02% sodium azide.
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9
ASSAY PREPARATION
10.2
Preparation of suspension cells
10.2.1 To ensure efficient cross-linking of the suspension
cells to the amine plate, cells must be grown and
treated in a different plate or dish of choice. The cell
seeding density of the amine plate is cell typedependent. As an example, HL-60 and Jurkat cells
should be seeded, respectively, at 300,000 and
200,000 cells per well in 100 µLof media.
10.2.2 When treatment is completed, transfer treated
suspension cells to the amine plate.
10.2.3 Centrifuge the microtiter plate at 500 x g for 5-10
minutes.
10.2.4 Fix cells by adding a final concentration of 4%
Paraformaldehyde Solution. This can be achieved
by overlaying an equal volume of 8%
Paraformaldehyde Solution to the culture volume
(e.g. add 100 µL 8% Paraformaldehyde to a well
with 100 µL media).
10.2.5 Centrifuge the microtiter plate again at 500 x g for
5-10 minutes.
10.2.6 Continue in the fixation for a total of 15 - 20 minutes.
Note – Both paraformaldehyde and sodium azide
are toxic, handle with care and dispose of according
to local regulations.
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10
ASSAY PROCEDURE
11. ASSAY PROCEDURE
●
It is recommended to use a plate shaker (~200 rpm) during all
incubation steps. Any step involving removal of buffer or
solution should be followed by blotting the plate gently
upside down on a paper towel before refilling wells. Unless
otherwise noted, incubate at room temperature.
●
It is recommended to assay all and samples in duplicate.
11.1 Remove 1X PBS and add 200 µL of pre-heated Antigen
Retrieval Buffer (see 9.4) to each well of the plate. Incubate
for 15 minutes at 80⁰C.
11.2 Remove the Antigen Retrieval Buffer and wash the plate
three times briefly with 1X PBS. For each wash, rinse each
well of the plate with 200 µL.
11.3 Remove 1X PBS and add 200 µL of 1X Permeabilization &
Blocking Solution to each well of the plate. Incubate for
2 hours at room temperature.
11.4 Remove Permeabilization/Blocking Solution and add 100 µL
1X Primary Antibody Cocktail Solution to each well of the
plate. Incubate for 2 hours at room temperature or overnight
at 4°C.
Note – To determine the background signal it is essential to
omit primary antibody from at least one well containing cells
for each experimental condition.
11.5 Remove Primary Antibody Cocktail Solution and wash the
plate three times briefly with 1X Wash Buffer. For each
wash, rinse each well of the plate with 200 µL of
1X Wash Buffer. Do not remove the last wash until step
11.8.
11.6 Remove 1X Wash Buffer and add 100 µL 1X Secondary
Antibody Solution to each well of the plate. Incubate 2 hours
at room temperature in the dark.
11.7
Remove 1X Secondary Antibody Cocktail Solution and
wash five times briefly with 1X Wash Buffer. For each
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11
ASSAY PROCEDURE
wash, rinse each well of the plate with 200 µL of 1X Wash
Buffer.
Do not remove the last wash.
11.8
Wipe the bottom of the plate and the scanner surface with a
damp lint-free cloth to clean before scanning the plate on
the LI-COR® Odyssey® system. Collect data in the 700
and 800 channels according to manufacturer’s instructions.
The optimal focus off-set for typical amine plates is 3.9. The
Total mTOR protein signal corresponds to the 800 channel
(IRDye800®) and the mTOR (pSer2448) protein signal
corresponds to the 700 channel (IRDye680®).
Note – The absolute value of the IR signal is dependent on
the intensity settings. Value of 6.5 for the 700 and 800
channels is recommended for initial scanning. Adjust as
needed so that the signal is not saturated in any well.
11.9
Remove the last wash and add 100 µL of Janus Green
Stain to each well of the plate. Incubate plate for 10
minutes at room temperature.
Note – The IR signal should be normalized to the Janus
Green staining intensity to account for differences in cell
seeding density.
11.10 Remove the dye and wash the plate five times in deionized
water or until excess dye is removed.
11.11 Remove last water wash, blot to dry, add 100 µL of
0.5 M HCl to each well of the plate and incubate for
10 minutes in a plate shaker.
11.12 Measure OD595 nm using a standard microplate
spectrophotometer or measure a signal in the 800 nm
channel using a LI-COR® Odyssey® scanner.
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12
DATA ANALYSIS
12. CALCULATIONS
12.1 Background Subtraction
Determine the raw signal intensity (Integrated Intensity)
values for the IR700 and IR800 channels for the wells that
lacked primary antibody. Subtract the mean background
values from all other IR700 or IR800 experimental values
respectively.
12.2 Janus Green normalization
Divide the background subtracted IR intensities (from 12.1)
by the Janus Green value of the corresponding well. The
result is the “normalized intensity”.
12.3 Normalization of Phospho signal to total protein.
Divide the pSer2448 mTOR normalized intensity by the total
mTOR normalized intensity.
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13
DATA ANALYSIS
13. TYPICAL DATA
TYPICAL STANDARD CURVE – Data provided for demonstration
purposes only.
Assay performance was tested using HeLa cells treated with 10 nM
calyculin A for 15 minutes in 0F-HGDMEM media after overnight
serum starvation. Calyculin A phosphorylates mTOR at Ser2448.
Figure 1 shows dynamic range of the assay on amine coated plates.
Linearity of signal is observed between 25 – 100k per well.
mTOR Total
mTOR pSer2448
Treatment
Cells/Well
Calyculin A
Calyculin A
0
1562.5
3125
6250
12500
25000
50000
100000
0
0.33
0.37
0.72
2.07
3.03
6.08
9.96
0
-0.14
0.20
0.39
2.36
9.08
19.82
30.16
Figure 1. Dynamic range of mTOR total and pSer2448. Levels of total
mTOR and phosphorylated protein at Ser2448 were measured in calyculin A
treated cells. Serum-starved HeLa cells were seeded on amine coated plates
within the working range of the assay and treated with 10nM calyculin A in
serum free media for 15 minutes the day of fixation. The relative levels of Total
mTOR (Left) and mTOR pSer2448 (Right) are shown after background
subtraction.
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14
DATA ANALYSIS
14. TYPICAL SAMPLE VALUES
PRECISION –
The coefficient of the intra-assay variation for this assay kit on HeLa
cells is typically 5.71% for total and 3.32% for pSer2448. The assay
was also found to be highly robust with a mean Z factor from multiple
cell densities of 0.508 for mTOR (25k – 100k/well) and 0.800 for
mTOR pSer2448 (25 – 100k/well).
15. SPECIES REACTIVITY
This kit detects mTOR total protein and 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 2 shows performance of this kit on NIH3T3 PDGF treated
against BSA control
Figure 2. Mouse Reactivity of mTOR total and pSer2448 ICE assay. Levels
of total mTOR and phosphorylated protein at Ser2448 were measured in
serum-starved NIH3T3 cells that were treated with PDGF recombinant protein
at 50 ng/mL in 1% BSA. The relative levels of Total mTOR (left) and mTOR
pSer2448 (middle) are shown after background subtraction. Specific
phosphorylation was calculated by determining the ratio of pSer2448 to Total
mTOR levels after normalization (right).
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15
DATA ANALYSIS
16. ASSAY SPECIFICITY
Confidence in antibody specificity is critical to In-Cell ELISA data
interpretation; therefore the primary antibodies in this kit were validated
by In-Cell ELISA and Immunocytochemistry with the use of lambda
phosphatase and by Western blot with the presence of signal at the
correct band size.
mTOR pSer2448 was phosphorylated with calyculin A treatment,
followed by an artificial dephosphorylation with the use of lambda
phosphatase (LPP) on the ICE and ICC platform after fixation and
permeabilization and on western blot after cell lysis with a non-ionic
detergent but before the addition of SDS-PAGE buffer.
Figure 3. Specificity of Signal by In-Cell Elisa. Levels of total mTOR and
phosphorylated protein at Ser2448 were measured in DMSO and calyculin A
treated cells. Serum-starved HeLa cells were seeded on amine coated plates
within the working range of the assay and treated with 10 nM calyculin A or
DMSO control in serum free media for 15 minutes the day of fixation.
Specificity of phosphorylation was determined by treating calyculin A-treated
HeLa cells with LPP for 45 minutes at 40⁰C immediately after permeabilization.
The relative levels of Total mTOR (Left) and mTOR pSer2448 (Middle) are
shown after background subtraction. Specific Phosphorylation was calculated
by determining the ratio of pSer2448 to Total mTOR levels after normalization
(Right).
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16
DATA ANALYSIS
Figure 4. Specificity of Signal by Immunocytochemistry. HeLa cells were
seeded on glass coverslips and allowed to adhere overnight. After adhesion,
cells were treated with DMSO or calyculin A for 15 minutes and fixed with 4%
paraformaldehyde. The levels of mTOR total and mTOR pSer2448 were
measured following the protocol as described above until step 11.5. The
mouse mTOR total protein was labeled with goat anti-mouse-488 whereas the
rabbit mTORpSer2448 was labeled with goat anti-rabbit-594. mTOR total and
mTOR pSer2448 show specific co-localization in the cytoplasm.
The
cytoplasmic signal for the pSer2448 target is low on serum starved HeLa cells
treated with DMSO only (A), whereas it is significantly induced with the use of
10 nM calyculin A (B). The pSer2448 specific signal is completely removed
when calyculin A-treated HeLa cells are treated with LPP (C).
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17
DATA ANALYSIS
Figure 5. Verification of antibodies by Western Blot. Calyculin A-treated
HeLa Lysates were treated with λ Ppase (LPP) 34˚C or left untreated on ice.
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 antibody mTOR pSer2448 or the antibody
against total mTOR in 10% Blocking Buffer 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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18
RESOURCES
FREQUENTLY ASKED QUESTIONS
How many cells do I seed per well?
The cell seeding density varies by cell type and depends both on the
cell size and the abundance of the target protein. The cell seeding will
likely need to be determined experimentally by microscopic cell density
observation of serially diluted cells. For adherent cells, prepare a serial
dilution of the cells in a plate and allow them to attach prior to
observation. The goal is to have cells that are just confluent at the
time of fixation. Overly confluent cells may have compromised viability
and tend to not adhere as well to the plate. Under-seeded cells may
yield too low a signal, depending on the analyte. Keep in mind that
drug treatments or culture conditions may affect cell density/growth.
Do I have to use an amine-coated microplate?
We have tested black wall amine and cell culture treated microplates
and found that amine coated plates improve reproducibility and
specificity in comparison to standard plates. In addition, multiple cell
types appear to have the most favorable growth and even seeding on
amine plates. The assay performance is only guaranteed with amine
plates.
A treatment causes cells detachment. Is there a way to prevent the lost
of detaching cells?
Loss of floating cells can be easily prevented by inserting two
centrifugation steps into the protocol: (1) Immediately prior the addition
of Paraformaldehyde Solution (step 10.3) centrifuge the microtiter plate
at 500 x g for 5-10 minutes, (2) Immediately after the addition of
Paraformaldehyde Solution centrifuge the microtiter plate again at
500 x g for 5-10 minutes. Continue in the fixation for a total of 15 - 20
minutes. For examples using detaching cells in In-Cell ELISA, refer to
ab110215 Product Booklet.
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19
RESOURCES
I grow my cells in 15% FBS, will this interfere with the cell fixation?
Culture media containing up to 15% fetal serum does not interfere with
the cell fixation and cross-linking to the plate on adherent cells.
However we recommend for suspension cells to use no more than
10% fetal serum.
How do I measure the assay background?
It is essential to omit primary antibody in at least one well (3 wells
recommended) to provide a background signal for the experiment
which can be subtracted from all measured data. This should be done
for each experimental condition. It is also recommended to include at
least one well without cells to provide primary antibody background.
Is Janus Green normalization necessary?
Janus Green is a whole-cell stain that is useful to determine if a
decrease in IR intensity in a well is due to a relevant down-regulation
or degradation of the target analyte or if it is a function of decreased
cell number (e.g. due to cytotoxic effect of a treatment). As such it is
not a required readout, but it is useful in the analysis to determine a
normalized intensity value.
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RESOURCES
17. TROUBLESHOOTING
Problem
Cause
Solution
Too brief incubation
times
Ensure sufficient incubation
times
Inadequate reagent
volumes or improper
dilution
Check pipettes and ensure
correct preparation
Insufficient cells
Increase seeding density of
cells; goal is newly
confluent cells at time of
fixation.
Cell detachment
Refer to section 17
Plate is insufficiently
washed
Review the manual for
proper washing. If using a
plate washer, check that all
ports are free from
obstruction
Contaminated wash
buffer
Prepare fresh wash buffer
Artifacts creating
increased signal on
IR
Troughs used for
multichannel pipetting could
be dirty.
Edge effects
Do not use the edges of the
plate. Incubate in a humid
box
Variable cell seeding
Plate cells with care and
normalize with Janus Green
Low Signal
High CV
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21
RESOURCES
18. NOTES
LI-COR®, Odyssey®, Aerius®, IRDye®™ and In-Cell Western™ are registered trademarks or trademarks of LI-COR
Biosciences Inc
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22
RESOURCES
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23
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All information / detail is correct at time of going to print.
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