ab154468 –
AMPK alpha pThr172
ELISA Kit
Instructions for Use
For the measurement levels of AMPK alpha
phosphorylated at threonine 172 in human,
mouse and rat cell and tissue lysates.
This product is for research use only and is not
intended for diagnostic use.
Table of Contents
1.
Introduction
2
2.
Assay Summary
5
3.
Kit Contents
6
4.
Storage and Handling
6
5.
Additional Materials Required
7
6.
Preparation of Reagents
8
7.
Sample Preparation
9
8.
Assay Procedure
12
9.
Data Analysis
14
10. Specificity
18
11. Cross-reactivity
21
11. Troubleshooting
22
1
1. Introduction
Principle: ab154468 5'-AMP-activated protein kinase catalytic
subunit alpha (AMPKα1, PRKAA1) phospho T172 ELISA (EnzymeLinked Immunosorbent Assay) kit is an in-vitro enzyme-linked
immunosorbent assay for the accurate measurement of AMPK α
phospho T172 in human, rat and mouse lysates.
The assay
employs a specific mouse monoclonal antibody bound to the plate.
Standards and samples are pipetted into the wells in conjunction
with a rabbit monoclonal primary detector antibody. After washing
away excess protein and unbound detector antibody, an HRPconjugated secondary detector antibody (HRP Label) specific for the
primary detector antibody is pipetted to the wells. The wells are
again washed, a TMB substrate solution is added to the wells and
color develops in proportion to the amount of AMPKα pT172 bound.
The developing blue color is measured at 600 nm. Optionally, the
reaction can be stopped by adding hydrochloric acid which changes
the color from blue to yellow and the intensity can be measured at
450 nm.
2
Background: AMP-activated protein kinase (AMPK) is an energy
sensor protein kinase that plays a key role in regulating cellular
energy homeostasis. Mammalian AMPK is a heterotrimer kinase,
containing a catalytic subunit (α) and two regulatory subunits (β and
γ). Each subunit has different isoforms (α1, α2, β1, β2, γ1, γ2, γ3)
with
differential
tissue
expression,
cellular
localization
and
functionality. Human AMPK has a 98.7% sequence similarity with
mouse AMPK and a 99% sequence similarity with rat AMPK. It has
been hypothesized that when ADP or AMP are present at high
levels, these nucleotides bind directly to the γ subunit, leading to a
conformational change that allows phosphorylation of Thr172 at the
α subunit. Phosphorylation of AMPK α activates the kinase, which
leads to downstream effects concerted to increase catabolic
pathways and suppress anabolic pathways in order to restore levels
of cellular ATP and ultimately control cell fate.
AMPK is activated physiologically due to stresses such as low
nutrients and prolonged exercise.
activated
pharmacologically
by
Furthermore AMPK may be
metformin
(the
most
widely
prescribed Type 2 diabetes drug), phenformin, AICAR and
resveratrol.
Activation of AMPK due to low nutrients leads to
suppression of the mammalian target of rapamycin complex 1
(mTORC1) pathway allowing coordination and control of cell growth
and autophagy. Furthermore, AMPK also controls metabolism via
direct phosphorylation of metabolic enzymes such as Acetyl-CoA
carboxylase (ACC1 and ACC2) HMG-CoA reductase, hormone
3
sensitive lipase (HSL), adipose triglyceride lipase (ATGL), Insulin
receptor substrate 1 (IRS1) and phosphofructo-kinase (PFKFB).
Control of metabolism also occurs long term through control of
transcription and chromatin structure via phosphorylation of
transcription factors (SREBP1, PPARγ), co-activators (CRTC family,
PGC1α),
acetyltransferase
p300,
histone
H2B
and
histone
deacetylases (HDACs class IIa). Activation of AMPK has also been
linked to circadian clock regulation via phosphorylation of Cry1,
coupling daily light and dark cycles to the metabolic control of fed
and fasting cycles. In addition, it has been suggested that AMPK
may control cell polarity and cytoskeletal dynamics in some settings.
4
2. Assay Summary
Equilibrate all reagents to room temperature. Prepare all the
reagents, samples, and standards as instructed.
Add 25 µL of 2X sample to each well used and overlay with 25 µL of
2X detector antibody.
Incubate 3 hours at room temperature.
Aspirate and wash each well three times. Add 50 μL of prepared
HRP labeled secondary detector antibody. Incubate 1 hour at
room temperature.
Aspirate and wash each well four times. Add 100 μL TMB
Development Solution to each well. Immediately begin
recording the color development with elapsed time at 600 nm for
15 minutes. Alternatively add 100 μL of a Stop solution (1N HCl)
at a user-defined time and read at 450 nm.
5
3. Kit Contents
Item
Quantity
10X Wash Buffer
40 mL
Extraction Buffer
15 mL
10X Blocking Buffer
6 mL
TMB Development Solution
12 mL
20X AMPK alpha pT172 Detector Antibody
250 µL
10X HRP Label
AMPK α Microplate
1 mL
96 Wells
4. Storage and Handling
Store all components at 4°C (do not freeze the lyophilized protein).
This kit is stable for 6 months from receipt. Unused microplate strips
should be returned to the pouch containing the desiccant and
resealed.
6
5. Additional Materials Required

Microplate reader capable of measuring absorbance at
600 nm (or 450 nm after addition of Stop Solution - not
supplied.

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

Stop Solution (optional) – 1N hydrochloric acid

Optional plate shaker for all incubation steps

Well plate cover or seals
7
6. Preparation of Reagents
6.1
Equilibrate all reagents to room temperature (18-25°C)
prior to use.
6.2
Prepare 1X Wash Buffer by adding 40 mL of 10X Wash
Buffer to 360 mL of nanopure water.
6.3
Prepare 1X Incubation Buffer by adding 6 mL of 10X
Blocking Buffer to 54 mL of 1X Wash Buffer.
Excess
unused 1X Incubation buffer may be stored at -20°C for 6
months after performing the ELISA.
6.4
Prepare 2X AMPK alpha pT172 primary detector antibody
with 1X Incubation Buffer immediately prior to use, by
diluting the supplied stock 10 fold. Prepare 250 µL for
each 8 well strip used.
6.5
Prepare 1X HRP labeled secondary antibody by diluting
the supplied stock 10-fold with 1X Incubation Buffer
immediately before use. Prepare 500 µL for each 8 well
strip used.
8
7. Sample Preparation
Note: The extraction buffer provided with this kit may be
supplemented with phosphatase inhibitors, PMSF and protease
inhibitor cocktail prior to use.
Supplements should be used
according to manufacturer’s instructions.
7.1 Preparation of extract from cell pellets (Human, mouse, rat).
7.1.1
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 10 min at 4°C.
7.1.2
Rinse cells twice with PBS.
7.1.3
Solubilize cell pellet at 4x107/mL in Extraction Buffer.
7.1.4
Incubate on ice for 20 minutes. Centrifuge at 15,000
x g, 4°C for 10 minutes. 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 may be quantified using
a protein assay.
7.2 Preparation of extracts from tissue homogenates.
7.2.1
Tissue
lysates
are
typically
prepared
by
homogenization of tissue that is first minced and
9
thoroughly rinsed in PBS to remove blood (dounce
homogenizer recommended).
7.2.2
Homogenize 100 to 200 mg of wet tissue in 500 µL
– 1mL of the supplied extraction buffer. For lower
amounts of tissue adjust volumes accordingly.
7.2.3
Incubate on ice for 20 minutes. Centrifuge at 10,000
x g, for 10 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 may be
quantified using a protein assay.
7.3 Preparation of dilution series of a positive control sample
Note: It is strongly recommended to prepare a dilution series of a
positive control sample. The levels of phosphorylated Threonine
172 of AMPK α are elevated in certain cell lines such as Hek293T
and may be relatively low in other cells and tissues. Use a
suitable positive control sample to prepare the dilution series. The
relative levels of phosphorylated Threonine 172 of AMPK α in
other experimental samples can be interpolated from within this
positive control sample series.
7.3.1
To prepare serially diluted positive control sample,
label six tubes #2-7.
10
7.3.2
Prepare a positive control sample lysate or extract
as directed in previous section (7.1). Dilute the
positive control sample to 2X of the upper
concentration limit of the working range of the assay
in 1X Incubation Buffer. The final volume should be
no less than 150 µL. Label this tube #1. As a guide,
typical
ranges
of
sample
concentration
for
commonly used sample types are shown below:
Typical working ranges
Sample Type
Range
Hek293T cells
5 – 200 µg/mL
oligomycin treated H4IIE
20 – 1000 µg/mL
Starved C2C12 myoblasts
10 – 1000 µg/mL
Phenformin treated MCF7 cells
20 – 1000 µg/mL
7.3.3
Add 75 µL of the Incubation Buffer to each of tubes
#2 through #7.
7.3.4
Transfer 75 µL from tube #1 to tube #2. Mix
thoroughly. With a fresh pipette tip transfer 75 L
from #2 to #3. Mix thoroughly. Repeat for Tubes #4
through #7. Use the diluent as the zero standard
11
tube labeled #8. Use fresh control sample dilution
series for each assay.
75l
75l
75l
75l
75l
75l
1
Positive
control
sample
2
3
4
5
6
7
8
1/2
1/4
1/8
1/16
1/32
1/64
Blank
75L
8 Assay Procedure
Equilibrate all reagents to room temperature before use. It is
recommended all samples and standards be assayed in
duplicate. If available use a plate shaker for all incubation steps
at 300rpm.
8.1
Equilibrate all reagents, and samples as directed in the
previous sections.
8.2
Remove unused microplate strips from the plate frame,
return them to the foil pouch containing the desiccant
pack, and reseal.
12
8.3
The samples should be diluted at a 2X concentration
within the working range of the assay in 1X Incubation
Buffer. As a guide, typical ranges of sample concentration
are shown above (section 7.3.2).
Add 25 µL of each
sample at 2X concentration. Also include a 1X Incubation
buffer as a background control.
8.4
Overlay 25 µL 2X Primary Detector antibody in incubation
buffer (step 6.4) to the sample. Cover/seal the plate and
incubate for 3 hours at room temperature.
8.5
Aspirate each well and wash, repeat this twice more for a
total of three washes. Wash by aspirating or decanting
from wells then dispensing 300 µL 1X Wash buffer into
each well.
Complete removal of liquid at each step is
essential to good performance.
After the last wash,
remove the remaining buffer by aspiration or decanting.
Invert the plate and blot it against clean paper towels to
remove excess liquid.
8.6
Add 50 µL 1X HRP labeled secondary detector antibody in
1X incubation buffer (step 6.5) to each well used.
Cover/seal the plate and incubate for 1 hour at room
temperature.
8.7
Repeat the aspirate/wash procedure above.
13
8.8
Add 100 µL TMB Development Solution to each empty
well and immediately record the blue color development
with time in the microplate reader prepared with the
following settings:
Mode:
Kinetic
Wavelength:
600 nM
Time:
up to 15 min.
Interval:
20 sec. - 1 min.
Shaking:
Shake between readings
Alternative– In place of a kinetic reading, at a user defined
time, record the endpoint OD data at (i) 600 nm or (ii) stop
the reaction by adding 100 µL stop solution (1N HCl) to each
well and record the OD at 450 nm. Analyze the data as
described below.
9. Data Analysis
Subtract average zero 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
14
(e.g. linear, semi-log, log/log, 5 parameter logistic). Read relative
AMPK α phospho T172 concentrations for unknown samples from
the curve plotted. Samples producing signals greater than that of
the highest control curve should be further diluted in 1X Incubation
buffer and reanalyzed, then multiplying the concentration found by
the appropriate dilution factor.
TYPICAL POSITIVE SAMPLE RANGE - For demonstration only.
mOD/min (600nm)
100
10
1
1
10
100
Hek293T Lysate [g/mL]
Figure 1. Example of positive control sample standard curve. A dilution
series of extract in Incubation Buffer in the working range of the assay. The
extract was prepared with Hek293T cells grown in High Glucose DMEM
supplemented with 10% FCS.
15
SENSITIVITY
Calculated minimum detectable dose = 4.5 µg/mL (zero dose n=8 +
2 standard deviations) using Hek293T cells
LINEARITY OF DILUTION
Linearity of dilution was determined by comparing dilution series of
extracts prepared from Hek293T cells (starting protein concentration
is 100 µg/mL) to extracts prepared from serum starved C2C12 cells
and Oligomycin treated H4IIE cells
Dilution Factor
C2C12 dilution [100
H4IIE dilution [500
µg/mL] % Expected
µg/mL] % Expected
value
Value
1:1
100%
100%
1:2
90%
89%
1:4
87%
116%
1:8
92%
124%
1:16
112%
129%
16
RECOVERY
Sample Type
Average Recovery (%)
Range (%)
72%
58 – 84%
63.3%
52 – 74%
83%
73 – 94%
50% Culture media
(8 dilutions)
10% Serum
(8 dilutions)
50% Extraction
buffer
(8 dilutions)
Since interference is observed, for more accurate comparison dilute
all samples in equivalent buffers/media/supplements
REPRODUCIBILITY
Parameter
CV%
Intra (n= 22)
5.6%
Inter (n=3)
5.3%
17
10. Specificity
The specificity of the assay to measure AMPK α phosphorylated at
threonine 172 was demonstrated with the use of λ protein
phosphatase (λ Ppase) treatment of Hek293T extracts. The relative
levels of the phosphorylated T172 in extracts decreased dramatically
when treated with 1/100 dilution of the enzyme (Fig. 2). This result
matched well with a parallel Western blot analysis (using the kit’s
detector antibody) of the same  protein phosphatase treated
sample used on the ELISA format.
The total levels of AMPK α
protein were not sensitive to  protein phosphatase treatment as
determined by Western blotting (Fig. 3).
Hek293T cells 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). λ Ppase 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.
18
mOD/min (600nm)
~150ug/mL loading
80
60
40
20
P
ek
29
3T
H
H
ek
29
3T
pa
se
0
Figure 2. The AMPK α pT172 ELISA specifically measures the
phosphorylated threonine. Hek293T extracts were left untreated (control),
treated with 1:100 dilution of λ Ppase at 34˚C. Samples were loaded at 150
µg/mL on the plate and measured following the kit’s protocol. Treatment of
Hek293T extracts with λ Ppase decreases the signal to background levels
19
Figure 3. Western blot using capture anti-AMPK α (left) and detector
anti-AMKPK α pT172 (right) antibody. The detector antibody used in this
kit specifically detects the phosphorylated AMPK α as determined by
Western blotting. Hek293T extracts were treated with 1:100 dilution of λ
Ppase at 34˚C (lane 1) or untreated (lane 2). Samples were then diluted in
SDS-PAGE buffer and loaded at 40 µg/well. Membranes were blocked with
2X blocking buffer (ab126587) diluted in PBS for 1 hour and incubated with
either the capture antibody against total AMPK α (Left) or the detector
antibody AMPK α phospho T172 (Right) in 1X blocking buffer (ab126587)
diluted in PBS/0.05% Tween-20 overnight. Labeling was carried out with
secondary
antibodies
conjugated
to
HRP.
λ
Ppase
completely
dephosphorylates AMPK α without affecting the protein levels.
20
11. Cross-reactivity
100
mOD/min 600nm
mOD/min 600nm
100
10
1
1
10
100
Serum starved
C2C12 myoblasts [g/mL]
1000
10
1
0.1
1
10
100
1000
Oligomycin treated
H4IIE [g/mL]
Figure 4. AMPK α1 ELISA assay - dynamic range in rodent cell lines.
Relative levels of AMPK α pT172 can be measured in rodent samples. Left
panel shows mOD/min data obtained with C2C12 (mouse myoblasts) serum
starved overnight in 5mM glucose DMEM media. Right panel shows H4IIE
(rat hepatocarcinoma) serum starved overnight in 5 mM glucose DMEM
media and treated with 1 µM oligomycin for 3 hours.
21
11. Troubleshooting
Problem
Cause
Incubation times too
brief
Low Signal
Inadequate reagent
volumes or improper
dilution
Check pipettes and ensure
correct preparation
Plate is insufficiently
washed
Review manual for proper
wash technique. If using a
plate washer, check all ports
for obstructions
Contaminated wash
buffer
Make fresh wash buffer
Improper storage of
the ELISA kit
Store components at 4°C.
Keep substrate solution
protected from light.
Large CV
Low sensitivity
Solution
Ensure sufficient incubation
times; change to overnight
standard/sample incubation
22
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All information / detail is correct at time of going to print.
23