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Qian Gao, Kris Simonyi, and Claudia Suen, Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Drive, Hercules, CA 94547 USA
Introduction
Western blotting is a traditional technique for detecting phosphorylated
proteins within a variety of cell culture and tissue sample lysates. In
recent years, new techniques that apply a sandwich antibody capture approach
have become available for detecting phosphorylated proteins. These techniques
include Bio-Plex phosphoprotein assays, which are multiplex bead-based
assays for the detection of one or more different phosphorylated proteins
within a single sample. Enzyme-linked immunosorbent assays (ELISA) are
another technique that has been increasingly applied to detect phosphorylated
proteins a single target at a time. In this study, commercially available
Bio-Plex phosphoprotein and total target assays, and phosphoprotein and
total ELISA assays, are compared with western blotting in terms of sensitivity,
detection range, and level of correlation, for the detection of phospho-
and total ERK and p38 MAPK.
Methods
Lysate Preparation
Lysates of HEK 293 and HeLa cells were prepared according to two techniques.
The Bio-Plex cell lysis kit was used to prepare cell lysates for western
blot and Bio-Plex analysis (refer to bulletin 3033). A different lysing
protocol specified in the ELISA kit vendors manual was followed to prepare
cell lysates for ELISA.
Assay Procedure
The procedure detailed in bulletin 3033 was followed for western blot
analysis. The procedure provided in the Bio-Plex phosphoprotein detection
reagent kit was followed for the Bio-Plex phosphoprotein and total target
assays (summarized in the flowchart opposite). The procedure provided
in the ELISA kit vendors manual was followed for the phospho- and total
protein ELISAs. The same antibodies specific to each target were used
in both western blot and Bio-Plex phosphoprotein and total target assays.
The antibodies used in the ELISAs were those provided in the vendor kit.
Results and Discussion
Total p38 MAPK
Total p38 MAPK was detected by ELISA in HEK 293 lysates with protein concentration
ranging from 20 g/well to 0.625 g/well and absorbance ranging from 2.623
to 0.317 (Figure 1A). The limit of detection appears to be around 0.625
g/well. A similar dose response was observed with the Bio-Plex assay;
however, an MFI value of 897 at the lowest data point (0.625 g/well)
suggests the potential to detect further-diluted samples (Figure 1B).
The data also suggest high-end saturation over 10 g/well. With the western
blot, the target was detected down to a protein concentration of 1.25
g/lane (Figure 1C).
Total p38 MAPK was not detected, nor was a dose response observed with ELISA of HeLa lysates, even at the highest protein concentration tested (Figure 1D). This target was, however, detected with the Bio-Plex assay in each sample, with the MFI ranging from 4,950 to 321 (Figure 1E), which correlated well with the western blot (Figure 1F).
Phospho-p38 MAPK
Phospho-p38 MAPK (Thr180/Tyr182) was detected by ELISA in UV-treated HEK
293 lysate with protein concentration ranging from 12 g/well to 0.75
g/well (Figure 2A). The ELISA displayed a high-end saturation plateau
in samples with a protein concentration from 12 g/well to 3 g/well (Figure
2A), which suggests that further dilution is necessary to detect a dose
response. The Bio-Plex assay showed a distinct dose response (Figure 2B)
that correlated well with the western blot (Figure 2C).
Total ERK
Total ERK1/2 was detected by ELISA in HEK 293 lysates with protein concentration
ranging from 10 g/well to 0.625 g/well and absorbance ranging from 0.331
to 0.160 (Figure 3A). To explore the Bio-Plex total ERK2 assay sensitivity
further, lysates were further diluted to a protein concentration range
of 1.250.078 g/well (Figure 3B). In comparison, a faint band was visible
on the western blot at a total protein concentration of 0.625 g/lane
(Figure 3C).
Both sandwich antibody methods detected total ERK in HeLa lysates with protein concentration ranging from 13 g/well to 0.41 g/well. A flattened lower-end dose response was observed with the ELISA, and an absorbance range of 0.5000.158 showed a limited ability to distinguish between different samples (Figure 3D). The Bio-Plex total ERK2 assay better distinguished signal intensities from the high end to the low end, with MFI from 11,713 to 603 (Figure 3E). The western blot band became faint at 3.25 g/well, indicating the level of sensitivity compared to the Bio-Plex assay (Figure 3F).
Phospho-ERK
Epidermal growth factor (EGF)-treated HEK 293 cell lysates were used for
phospho-ERK (Thr202/Tyr204) assay comparison. Lysates were prepared using
the Bio-Plex cell lysis kit or by following a protocol provided with the
ELISA kit. ELISA showed a limited ability to distinguish the first two
samples, with total protein concentrations of 2 g/well and 1 g/well;
the reported sample absorbance reached a plateau, and the highest OD reported
was only 1.755 (Figure 4A). The Bio-Plex assay demonstrated distinctly
separate signals across all the samples (Figure 4B), which correlated
well with the western blot bands (Figure 4C).
Conclusions
For the detection of total p38 MAPK in HEK 293 cells, both the Bio-Plex
assay and ELISA correlated well with the western blot. The Bio-Plex assay
showed saturation at the high end but potential to detect the target in
further-diluted samples. With the HeLa lysate, the Bio-Plex assay detected
the target with good sensitivity and demonstrated a broad detection range
that correlated well with the western blot bands. The target was not detectable
by ELISA
For the detection of phospho-p38 MAPK in HEK 293 cells, the Bio-Plex assay demonstrated a broad detection range at the high end and good correlation with the western blot. Bio-Plex and ELISA assays both showed potential for higher sensitivity toward more dilute samples
For the detection of total ERK, the Bio-Plex assay demonstrated better signal distinction and sensitivity compared to ELISA
For phospho-ERK detection, the ELISA showed saturation at the high end. The Bio-Plex assay showed better signal distinction, dose response, and correlation with western blots compared to ELISA
The Bio-Plex suspension array system includes fluorescently labeled microspheres
and instrumentation licensed to Bio-Rad Laboratories, Inc. by the Luminex
Corporation.
Information in this tech note was current as of the date of writing (2004) and not necessarily the date this version (Rev A, 2004) was published.
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