ab157397 –
AMPKa (pSer496)
Human ELISA Kit
Instructions for Use
For the quantitative measurement of phosphorylated Ser496 of
AMPKa protein in Human cell and tissue lysates.
This product is for research use only and is not intended for diagnostic
use.
Version 2 Last Updated 28 February 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
16. TYPICAL SAMPLE VALUES
17. ASSAY SPECIFICITY
14
16
17
RESOURCES
18. TROUBLESHOOTING
19. NOTES
20
21
INTRODUCTION
1.
BACKGROUND
Abcam’s AMPKa pSer496 Human ELISA (Enzyme-Linked
Immunosorbent Assay) kit is designed for the accurate quantitative
measurement of phosphorylated Ser496 of AMPKa protein in human
cell and tissue lysates.
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 (alpha) and two regulatory subunits (beta and gamma). Each
subunit has different isoforms (alpha 1, alpha 2, beta1, beta2,
gamma1, gamma2, gamma3) 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.
Phosphorylation of AMPKa at Serine 496 has been shown by rat
mutagenesis studies to affect the activation of 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 alpha subunit and
subsequent activation. Activation of AMPK leads to a concerted
downstream effect that ultimately increases catabolism while
suppresses anabolism to restore cellular ATP and control cell fate.
This is carried out by its kinase activity which controls downstream
targets important in metabolism, autophagy, transcription and
chromatin structure.
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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 standard or sample to each well
used.
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. Immediately begin recording the
color development. Alternatively add a
Stop solution at a user-defined time.
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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.
5. MATERIALS SUPPLIED
Amount
Storage
Condition
(Before
Preparation)
10X Wash Buffer
Extraction Buffer
40 mL
15 mL
4ºC
4ºC
10X Blocking Buffer
AMPKa Microplate (12 x 8 well strips)
20X AMPKa (pSer496) Detector Antibody
10X HRP Label
TMB Development Solution
6 mL
4ºC
4ºC
4ºC
4ºC
4ºC
Item
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96 wells
350 µL
1 mL
12 mL
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.

Stop solution (optional) – 1N Hydrochloric acid.

Plate shaker for all incubation steps (optional).

PMSF (or other protease inhibitors) and phosphatase inhibitors.
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.

The sample should be diluted to within the working range of the
assay in Incubation Buffer.

As a guide, typical ranges of sample concentration for commonly
used sample types are shown below in Sample Preparation
(section 11).
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 the extraction buffer with protease
phosphatase inhibitors immediately prior to use.
9.3
and
1X Supplemented Incubation Buffer
Prepare 1X 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 10mM Sodium
Fluoride. Mix gently and thoroughly.
9.4
1X Incubation Buffer
Prepare 1X Incubation Buffer by adding 1 mL 10X Blocking
Buffer to 9 mL 1X Wash Buffer. Mix gently and thoroughly.
9.5
2X AMPKa pSer496 Detector Antibody
Prepare 2X AMPKa pSer496 primary detector antibody with
1X Supplemented Incubation Buffer immediately prior to use,
by diluting the supplied stock 10 fold. Prepare 250 µL 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 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 Ser496 of AMPKa are elevated in certain cell
lines such as Hek293T and may be relatively low in other cells lines
like HeLa (see Specificity section below). In cell lines where levels of
phosphorylation are low, AMPKa Ser496 may be phosphorylated with
calyculin A treatment (e.g. HeLa cells treated with 50 nM calyculin for 3
hours in media containing 10% FCS). Use a positive control sample to
prepare the dilution series. The relative levels of AMPKa pSer496 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 of 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
Hek293T cells
5 – 500 µg/mL
Jurkat cells
5– 500 µg/mL
11.1 Preparation of extracts from cell lysates
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 supplemented with
phosphatase inhibitors.
11.1.3 Solubilize cell pellet at 4x107/mL in Supplemented
Extraction Buffer
11.1.4 Incubate on ice for 20 minutes. Centrifuge at
15,000 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. The sample protein concentration in
the extract may be quantified using a protein assay.
11.2 Preparation of extracts from adherent cells by direct
lysis (alternative protocol)
11.2.2 Remove growth media and rinse adherent cells 2
times in PBS supplemented with phosphatase
inhibitors.
11.2.3 Solubilize the cells by addition of Supplemented
Extraction Buffer directly to the plate (use
750 µL - 1.5 mL Supplemented Extraction Buffer per
confluent 15 cm diameter plate).
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9
ASSAY PREPARATION
11.2.4 Scrape the cells into a test tube and incubate the
lysate on ice for 15 minutes. Centrifuge at
15,000 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. The sample protein concentration in
the extract may be quantified using a protein assay.
11.3 Preparation of extracts from tissue lysates
11.3.2 Tissue lysates are typically prepared by
homogenization of tissue that is first minced and
thoroughly rinsed in PBS to remove blood (dounce
homogenizer recommended).
11.3.3 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.
11.3.4 Incubate on ice for 20 minutes. Centrifuge at
15,000 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.
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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 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
blanks, omitting sample 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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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.
●
If available use a plate shaker for all incubation steps at
400rpm.
13.1 Prepare all reagents, working standards, and samples as
directed in the previous sections.
13.2 Remove excess microplate strips from the plate frame,
return them to the foil pouch containing the desiccant pack,
and reseal.
13.3 The samples should be diluted at a 2X concentration within
the working range of the assay in 1X Supplemented
Incubation Buffer. As a guide, typical ranges of sample
concentration are shown in section 11. Add 25 µL of each
sample
at
2X
concentration.
Also
include
a
1X Supplemented Incubation Buffer as a background
control.
13.4 Overlay 25 µL of 2X Primary Detector antibody in Incubation
Buffer (step 9.5) to the sample. Cover/seal the plate and
incubate for 3 hours at room temperature.
13.5 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.
13.6 Add 50 µL 1X HRP Label in 1X Incubation Buffer (step 9.4)
to each well used. Cover/seal the plate and incubate for
1 hour at room temperature.
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ASSAY PROCEDURE
13.7 Repeat the aspirate/wash procedure above.
13.8 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:
10 sec. - 1 min.
Shake before and between
readings
Shaking:
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 50 µL Stop Solution to each well and
record the OD at 450 nm
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13
DATA ANALYSIS
14. CALCULATIONS
Average the duplicate standard reading for each standard, sample and
control blank. Subtract the control blank from all mean readings. Plot
the mean standard readings against their concentrations and 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). Extrapolate protein concentrations for unknown and control
samples from the standard curve plotted. Samples producing signals
greater than that of the highest standard should be further diluted in
appropriate buffer and reanalyzed, then multiplying the concentration
found by the appropriate dilution factor.
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14
DATA ANALYSIS
15. TYPICAL DATA
TYPICAL STANDARD CURVE
demonstration purposes only.
-
Data
provided
for
Standard Curve Measurements
O.D.
Conc
(µg/mL)
1
2
3
3.91
7.81
15.63
31.25
62.50
125.00
250.00
500.00
1.2
1.5
2.7
4.4
7.8
14.3
31.8
57.8
1.2
1.4
2.8
4.4
8.8
15.2
30.4
61.4
1.3
1.4
2.7
4.3
7.9
13.7
31.9
55.6
Mean
O.D.
1.2
1.5
2.7
4.4
8.2
14.4
31.3
58.2
Figure 1. A Standard curve and data (read at 600nm) 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 as described in section
11.1.
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15
DATA ANALYSIS
16. TYPICAL SAMPLE VALUES
SENSITIVITY Calculated minimum detectable dose = 3.1 µg/mL (zero dose n=16 + 2
standard deviations) using Hek293T cells.
RECOVERY –
(Sample spiking at a range of concentrations in representative sample
matrices)
Average %
Recovery
89.5
104.5
99.2
Sample Type
50% Culture Media (8 Dilutions)
10% Serum (8 Dilutions)
50% Extraction Buffer (8 Dilutions)
Range
72.1 – 102.1
92.9 - 119
89 – 104.4
LINEARITY OF DILUTION Dilution
1
1:2
1:4
1:8
1:16
Jurkat dilution (µg/mL)
% Expected Value
100
101
83
103
101
PRECISION –
n=
Average CV %
IntraAssay
22
2.5
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InterAssay
3
2.9
16
DATA ANALYSIS
17. ASSAY SPECIFICITY
The specificity of the assay to measure AMPKa phosphorylated at
Ser496 was demonstrated with the use of λ protein phosphatase
(λ Ppase) treatment of Hek293T extracts. The relative levels of the
phosphorylated Ser496 in extracts decreased dramatically when
treated with 1/100 dilution of the enzyme (Figure. 2). 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. 3).
Hek293T cells and HeLa cells treated with 50 nM of calyculin 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.
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17
DATA ANALYSIS
Figure 2. The AMPKa pSer496 ELISA specifically measures the
phosphorylated Serine.
Hek293T extracts were left untreated
(control), treated with 1:100 dilution of λ Ppase at 34˚C. Samples were
loaded at 100 µg/mL on the plate and measured following the kit’s
protocol. Treatment of Hek293T extracts with λ Ppase decreases the
signal 5 fold.
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18
DATA ANALYSIS
Figure 3. Western blot using capture anti-AMPKa and detector
anti-AMKPKa pSer496. The detector antibody used in this kit
specifically detects the phosphorylated AMPKa as determined by
Western blotting. Hek293T extracts and HeLa calyculin extracts were
treated with 1:100 dilution of λ Ppase at 34˚C or. Samples were then
diluted in SDS-PAGE buffer and loaded at 40 µg/well. Membranes
were blocked with 5% milk in TBST for 1 hour and incubated with
either the detector antibody AMPKa pSer496, the capture antibody
against total AMPKa or anti-actin antibody (ab8224) in 1% milk in
TBST overnight. Labeling was carried out with secondary antibodies
conjugated to HRP. Treatment with calyculin in HeLa cells upregulates
phosphorylation at Ser496 and λ Ppase completely dephosphorylates
AMPKa
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19
RESOURCES
18. TROUBLESHOOTING
Problem
Cause
Solution
Poor standard
curve
Inaccurate Pipetting
Check pipettes
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
Low Signal
Active phosphatses
in the extract
Incubation times with
TMB too brief
Large CV
Low sensitivity
Plate is insufficiently
washed
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
19. NOTES
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RESOURCES
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Japan
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All information / detail is correct at time of going to print.
RESOURCES
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