October 15, 2015 (Vol. 35, No. 18)
Efficient, High-Content Detection of Pathway Activiation
Phosphorylation is arguably the most important regulatory mechanism in biological systems, directing both the activity and subcellular localization of many key signaling proteins. Phosphorylation is often detected by isotope labeling or by 2D gel and mass spectrometry analyses, both highly sensitive methods.
However, because of the costly equipment and labor required for mass spectrometry, and the special isotope handling and disposal required for radiolabeling, phospho-specific antibodies are generally the more preferred tool for detection of protein phosphorylation.
ELISAs can be designed to rapidly and inexpensively detect phosphorylation-induced conformational changes in a specific protein, allowing many samples to be tested in parallel. Alternatively, phospho-specific antibodies can be multiplexed into an array format, allowing the simultaneous detection of multiple target phospho-proteins. Using these powerful tools, researchers can accomplish either high-throughput detection of a specific activated protein in multiple samples, or detection of phosphorylation signatures through parallel detection of many related phospho-proteins.
Assay Formats, Procedures, and Applications
The RayBio® Phosphorylation ELISAs are sandwich-based enzyme-linked immunosorbent assay kits that focus on a variety of key cellular processes such as Nf-κB signaling, AMPK signaling, autophagy, cell cycle, DNA damage repair, apoptosis, and insulin signaling. In each ELISA microplate, up to 24 different cell or tissue lysate samples may be simultaneously tested in duplicate, producing both phosphorylated and total protein signal outputs.
The assay protocol includes four simple steps: 1) incubation of lysates in an antibody-coated 96-well microplate, 2) incubation with anti-phosphorylated antibody or anti-pan antibody, 3) incubation with an HRP-conjugated secondary antibody, and 4) colorimetric detection of captured protein using TMB substrate solution. With minimal hands-on time, this assay format removes the burden of running multiple Western blots, making this assay ideal for researchers who want to quickly screen multiple time points, treatments, drug doses, or cell lines.
RayBio Phosphorylation Antibody Arrays are rapid and convenient tools for researchers wishing to discover specific pathways or receptors that are activated in their experimental model systems. This planar array-based system features a nitrocellulose membrane or glass slide solid support, each spotted with a panel of antibodies against selected classical signaling pathway proteins.
In this scenario, the cell or tissue lysate sample is added into the array membrane incubation tray or glass slide chamber, followed by washes and addition of biotinylated detection antibodies. Signal detection is accomplished with a streptavidin-conjugated HRP or fluor.
The RayBio Human RTK Phosphorylation Array is specifically designed for simultaneously identifying relative levels of phosphorylation of 58 different human receptor tyrosine kinases (RTKs) and 13 closely related proteins which are also regulated by tyrosine phosphorylation. RTKs are key regulators of critical cellular processes, including proliferation, differentiation, survival, metabolism, and migration. Aberrant activation of RTK signaling pathways has been causally linked to many cancers and other diseases, thus prompting the development of many new generation drugs which target RTK activity.
The RayBio Human EGFR Phosphorylation Antibody Array is specifically designed to focus on site-specific activation of the epidermal growth factor receptor (EGFR) family, which includes four family members: EGFR, ErbB2, ErbB3, and ErbB4. The EGFR is activated by binding of its specific ligands, which then stimulates the receptor’s intrinsic intracellular protein-tyrosine kinase activity. This autophosphorylation elicits downstream regulation of cellular process such as cell migration, adhesion and proliferation. RayBio Human EGFR Phosphorylation Antibody Array utilizes 17 phospho-specific antibodies targeting specific residues on the EGF receptor, allowing detection of a highly detailed phosphorylation signature.
Figure 1. The Human Phospho-RTK Array detects tyrosine-phosphorylated receptors in A431 cell lysates. Representative images of The Human Phospho-RTK Array applied with cell lysates of A431 cells untreated (A) or treated with 100 ng/mL recombinant human EGF for 10 minutes (B). 200 µg of lysate was run on each array. Quantitative results of pixel density of phosphorylation of EGFR, ErbB2, ErbB3, and PDGFRb are also shown (C). RTK Phosphorylation Array target map is shown in (D). POS=positive control (biotinylated IgG); NEG=negative control (buffer); Blank (no spot).
Here we present analyses of EGF activation using both RayBio phosphorylation arrays. In this study, A431 epidermoid carcinoma cells were treated with recombinant human epidermal growth factor (EGF) to induce EGFR-mediated signaling. Cell lysates were first analyzed by RayBio Human RTK Phosphorylation Antibody Array C1 (Figure 1).
With EGF stimulation, increased phosphorylation levels were observed in several proteins, including EGFR, EphA6, ErbB2, and PDGFRb, compared to untreated cell lysate. Spot densities were quantified to show relative levels of activation (Figure 1C). To determine which residues on the EGF receptors were responsive to EGF stimulation, we applied the same cell lysates to the RayBio human EGFR Phosphorylation Antibody Array. As shown in Figure 2, the array revealed phosphorylation at Tyr845, Tyr1173, Ser1070 and Tyr1086 of EGFR, and Tyr1112 and Tyr1248 of ErbB2 in the stimulated cells compared to the untreated cells.
To further validate individual data points from array experiments, or to analyze phosphorylation levels in a large number of samples, researchers can use Western blots or ELISA. Here we show an example of how the RayBio Phosphorylation ELISA kit can be used to measure phosphorylation levels of EGFR at Serine 1070 (Figure 3).
The same cell lysates were analyzed by both RayBio Human Phospho-EGFR (Ser 1070) ELISA kit (Figure 3A) and by Western blot (Figure 3B). Consistent with the array result, a robust phosphorylation of EGFR at Ser1070 was observed in parallel with total EGFR levels by both detection methods (Figure 3A&B). We note that immunoblots were incubated with the same anti-EGFR and anti-phospho-EGFR (Ser1070) used in the Raybio Phosphorylation ELISA kit.
Figure 2. The Human Phospho-EGFR Array detects tyrosine-phosphorylated receptors in A431 cell lysates. Representative images of the Human Phospho-EGFR Array applied with cell lysates of A431 cells untreated or treated with 100 ng/mL recombinant human EGF for 10 minutes (A). 200 µg of lysate was run on each array. Phospho-RTK Array target map is shown in (B). POS=positive control (biotinylated IgG); NEG=negative control (buffer); Blank (no spot).
Summary
Studying protein phosphorylation is essential for understanding regulatory mechanisms in biological systems, their implications in human disease, and for advancing therapeutics. RayBio Phosphorylation ELISA kits and Antibody Arrays are very convenient, rapid, and sensitive methods for monitoring protein phosphorylation.
With the phosphorylation ELISA, researchers can easily measure and compare phosphorylation levels of a specific residue of a specific protein among multiple samples or various experimental conditions. With phosphorylation array, researchers can quickly screen phosphorylation of multiple target proteins. Both methods represent more rapid and efficient alternative to multistep phospho-protein detection by immunoblot analysis, which requires a minimum of two days of processing time.
Figure 3. Detection of relative phospho-EGFR levels in A431 cells following stimulation with EGF. (A) Analysis of cell lysates using Human Phospho-EGFR (S1070) and pan EGFR ELISA kit (Cat# PEL-EGFR-S1070-T). (B) Immunoblot analysis of the lysates using antibodies against phospho-EGFR (S1070) and EGFR (the same antibodies used in the ELISA kit).
Hao Tang, Ph.D. ([email protected]), is R&D scientist and Ruo-Pan Huang, M.D., Ph.D., is CEO and founder at RayBiotech.
