Navigation Links
Detection of Phosphorylated and Total ERK and p38 MAPK by Bio-Plex Assay, ELISA, and Western Blotting, Rev A

Qian Gao, Kris Simonyi, and Claudia Suen, Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Drive, Hercules, CA 94547 USA

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.

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 t arget 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 fr om 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).

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).

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.

back to top


Page: All 1 2 3 4 5

Related biology technology :

1. Simple, Sensitive, and Rapid Detection of FLAG -Tagged Fusion Proteins
2. A New PCR-based Mycoplasma Detection Method
3. HSVision Molecular Beacon Detection Module Rapidly Detects Herpes Simplex Virus DNA
4. Detection and Identification of Phosphorylation Sites in Proteins Using LC/MS/MS with Neutral Fragment Loss Mapping
5. Detection of mRNAs on Cryosections of the Cardiovascular System Using DIG-Labeled RNA Probes
6. Gene Expression Arrays: Highly Sensitive Detection of Expression Patterns with Improved Tools for Target Amplification
7. The DIG System Nonradioactive and Highly Sensitive Detection of Nucleic Acids
8. Quantitative Measurement of Cell Proliferation Using the BrdU ELISA: A Comparison Between Colorimetric and Chemiluminescent Detection
9. Quantification of Nucleosomes in Serum by the Cell Death Detection ELISAplus
10. A Further Step in Understanding Apoptosis Direct Detection of PARP Cleavage
11. In Situ Cell Death Detection Kit
Post Your Comments:

(Date:11/25/2015)... November 25, 2015 Studies reveal ... human plaque and pave the way for more effective treatment ... cats     --> ... diagnosed health problems in cats, yet relatively little was understood ... collaborative studies have been conducted by researchers from the WALTHAM ...
(Date:11/25/2015)... 2015 /PRNewswire/ - Aeterna Zentaris Inc. (NASDAQ:  AEZS; TSX: ... prospects remain fundamentally strong and highlights the following ... received DSMB recommendation to continue the ZoptEC Phase ... the final interim efficacy and safety data ... men with heavily pretreated castration- and Taxane-resistant prostate ...
(Date:11/25/2015)... The Global Genomics Industry ... and in-depth study on the current state of ... ) , The report provides ... classifications, applications and industry chain structure. The Genomics ... including development trends, competitive landscape analysis, and key ...
(Date:11/24/2015)... N.J. (PRWEB) , ... November 24, 2015 , ... The ... the recipient of the 2016 USGA Green Section Award. Presented annually since 1961, the ... through his or her work with turfgrass. , Clarke, of Iselin, N.J., ...
Breaking Biology Technology:
(Date:10/29/2015)... 29, 2015 Daon, a global leader in ... released a new version of its IdentityX Platform ... North America have already installed IdentityX v4.0 ... a FIDO UAF certified server component as ... activate FIDO features. These customers include some of the ...
(Date:10/29/2015)... , Oct. 29, 2015  Connected health pioneer, ... the explosion of technology-enabled health and wellness, and the ... book, The Internet of Healthy Things ... or smartphones even existed, Dr. Kvedar, vice president, Connected ... health care delivery, moving care from the hospital or ...
(Date:10/29/2015)... , Oct. 29, 2015 Today, ... a partnership with 2XU, a global leader in ... a smart hat with advanced bio-sensing technology. The ... athletes to monitor key biometrics to improve overall ... partnership, the two companies will bring together the most ...
Breaking Biology News(10 mins):