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APPLICATION NOTE
Phospho-Protein Panel: AMMP® Assay versus Western Blot &
Electrochemiluminescence(ECL)
Phosphokinase Pathway Targeted in Protein
Research
The mitogen-activated protein (MAP) kinases are
evolutionarily conserved enzymes that play an important
role in modulating a variety of cellular processes, including
proliferation, differentiation and apoptosis. Four major
groups of MAP kinases have been identified in mammalian
cells, these are known as the extracellular signal-regulated
kinases (ERK 1 and 2), the c-Jun amino-terminal kinases
(JNK), the p38 kinases (p38) and ERK5. We looked at three
of the groups which are all important targets for cell cycle
regulation, apoptosis and cancer research.
P38 modulates the cell cycle, activation of DNA repair,
regulation of protein translation and initiation of the
immune response
ERK 1/2 are involved in the regulation of mitosis, meiosis
and control of cell proliferation in differentiated cells.
JNK/SAPK pathway is activated by stress (heat, irradiation)
and inflammatory and cytokine signals. JNK/SAPK is
an important marker in cell cycle regulation and cancer
research.
STUDY OBJECTIVE
This study was designed to compare the sensitivity and
assay development flexibility of the AMMP Assay versus
Westerns and electrochemiluminescence techniques.
Here we describe phosphorylated MAPK protein AMMP
assays and western blots for members of three of the MAP
kinase groups, p38, ERK 1/2 and JNK which are detected
in three different human and mouse whole cell lysates.
We have compared AMMP measurements to western
blots and estimated the lower limit of detection for each
analyte by both methods. We have analyzed each assay for
reproducibility, both within and between assays as well as
between days of analysis. Both AMMP assay and Western
results were then compared to published results for
ElectroChemiLuminescence (ECL).
Using the AMMP assay, a “best pairing” analysis was
done to identify the most effective antibody pairs. In the
complete study, assays for each phospho-protein were
developed in Jurkat, Hek293 and NIH3T3 cell lines. Cell
types and assay conditions were chosen to best match
the reported assay conditions of a commercially available
ECL phospho-protein multiplexed assay. Using the Jurkat
line data, this Application Note will illustrate the proof of
principle of the protein assays developed and verified in
less than 1 month.
AMMP® Phospho-Protein Assay Configuration
APPLICATION NOTE
THE ViBE® PLATFORM AND AMMP® ASSAY TECHNOLOGY - LOAD & GO
BioScale’s ViBE Workstation powered by AMMP technology enables a new
generation of protein analysis by providing highly sensitive and reproducible
detection and quantitation of analytes in complex matrices. Integrating simple
sample preparation and AMMP’s novel, automated, non-optical detection and
quantitation technology, the ViBE platform delivers the performance needed for
today’s research with the versatility to meet unique assay requirements. Whether it
is sensitive detection of key analytes or rapid assays for quick turnaround of results,
BioScale’s ViBE™ platform provides the sensitivity, range, speed, and versatility all in a
simple-to-use, walkaway bench top system.
MATERIALS
Commercially antibody pairs used
• Rabbit anti-p38 (whole molecule) and anti-pp38
•
Rabbit anti-SAPK/JNK (whole molecule) and anti-pSAPK/JNK
•
Rabbit anti-ERK1/2 (whole molecule) and anti-pERK1/2
Phospho-protein Antibody Pair Screening
The ViBE® Workstation is uniquely suited to allow efficient assay development of highly sensitive assays. Determining antibody pairs
for AMMP® immunoassays is a straightforward screening process on the ViBE Workstation as illustrated below. Many antibodies and
assay configurations can be probed within a microtiter plate to determine the best antibody pairs and configurations with which to
move forward into assay development.
Example: To establish the best antibody pairing for an AMMP phosphor-protein assay, phosphor-protein antibodies are screened by
testing antibody pairs in multiple cell lysate types known to contain the target phosphor-protein. The screening experiment reveals
the relative differences in signal from AMMP sandwich assay pairs in different configurations and in stimulated and unstimulated cell
lysates as well as assay performance with r-proteins or specific analytes and assay performance in negative controls. With sample prep
and run time on the ViBE, results were available within 8 hours.
R.U. (AMMP)
Best
Pairing
Anti-phospho
p38
2
Here we screened the following
commercial antibodies for p38
binding.
• Phospho-38 MAPK (Thr180/
Tyr182)(D3F9) XP Rabbit mAb
• Phospho-38 MAPK (Thr180/
Tyr182) Rabbit mAb
• p38 MAPK Antibody
• p38alpha MAPK Antibody
• Human/Mouse/Rat p38 alpha
Antibody
We tested duplicates of stimulated
cell lysates, recombinant protein, and
a negative control. Highly reactive
pairs were further tested with a bead
only control and the ones with the
greatest signal to noise difference
(highlighted in purple above) were
chosen.
APPLICATION NOTE
RESULTS
AMMP® pp38 Assay
AMMP® pp38 Assay Jurkat Cell Lysates
Electrochemiluminescence Multiplex Assay
Source: MesoScale Discovery Product Literature
The results of the p38 ViBE assay and
the western blot were compared for
detection limits in PMA/Calyculin
stimulated Jurkat cell lysates. The
AMMP assay signal was detectable
over background at 0.04 µg of pp38
while the western blot signal was
slightly visible to 0.075 µg. CVs for
the ViBE replicates were less than 16%
(n=3 replicates). Error bars on charts
indicated standard deviation between
replicates. Data from ECL analysis
for pp38 show signal detection over
background at 0.31 µg. Similar results
were seen in the HEK293 and 3T3 cell
lines.
Western Blot:
Anti-phospho p38 Jurkat Cell Lysates
AMMP® pERK Assay
AMMP® pERK Assay Jurkat Cell Lysates
Electrochemiluminescence Multiplex Assay
The results of the ERK 1/2 AMMP assay
and the western blot were compared
for detection limits in PMA/calyculin
stimulated Jurkat cell lysates. The
AMMP Assay signal was detectable over
background at 0.075 ug pERK while the
western blot signal was visible to 0.157
ug. CVs for the VIBE replicates were
less than 8%, (n=3 replicates). Data
from ECL analysis for pERK show signal
detection over background at 0.31 µg.
Similar results were seen in the HEK293
and 3T3 cell lines.
Source: MesoScale Discovery Product Literature
Western Blot:
Anti-phospho ERK 1/2 Jurkat Cell Lysates
3
Phospho-Protein Panel: AMMP™ Assay versus Western Blot & Electrochemiluminescence(ECL)
APPLICATION NOTE
AMMP® pJNK Assay
AMMP pJNK Assay Jurkat Cell Lysates
Electrochemiluminescence Multiplex Assay
The results of the JNK AMMP assay and the
western blot were compared for detection
limits in PMA/Calyculin stimulated Jurkat
cell lysates. The AMMP Assay signal was
detectable over background at 0.075 ug
while the western blot signal was visible to
0.313 ug. CVs for the VIBE replicates were
less than 10%, (n=3 replicates). Data from
ECL analysis for pJNK show signal detection
over background at 0.31 µg. Similar results
were seen in the HEK293 and 3T3 cell lines.
Western Blot:
Anti-phospho JNK Jurkat Cell Lysates
WORKFLOW: Significantly less hands-on-time with walkaway operation
Process workflow in a laboratory is greatly
enhanced using an AMMP assay. The assays
reduce the time to results , the amount of
manual technician time and the number of
error-producing sample handling steps. In fact,
in these assays technician hands-on time for
an AMMP assay is more than 8 times less than
western blot and an electrochemiluminescence
assays. With the AMMP Assay’s homogenous
format and the ViBE’s walkaway operation,
valuable technician time can be spent on more
critical laboratory needs.
CONCLUSIONS
As demonstrated the AMMP assay being a direct, homogeneous approach offers significant advantages in the speed and flexibility
of assay development. The three assays described here were developed in less than one week and optimized in three cell lines
in less than 2 months. The AMMP Assays have the ability to use off-the-shelf antibodies and reagents enabling the selection
of the most effective and specific antibody pairing and configuration for the analyte. All three phospho-proteins AMMP assays
showed increased sensitivity of at least 4X over the ECL Assay. AMMP Assay is far less labor intensive and time consuming than the
electrochemiluminescense and Western Blot assays.
APPNOTE–PhosphoP00120
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