Unveiling the complex interaction of cell signaling pathways
Thermo Fisher Scientific provides a variety of high-quality immunoassay solutions for cell signaling research. The offering covers convenient ELISA kits and spans to multiplex solution, including the novel ProcartaPlex Dual Reporter panels. These ProcartaPlex Dual Reporter assays enable the simultaneous detection and quantitation of the phosphorylated and total form of several proteins in a single well. The different assay formats provide the flexibility to study the various aspects of cell signaling pathways, including protein expression, phosphorylation, and activation.
Introducing the first dual reporter application for Luminex xMAP INTELLIFLEX DR-SE Instruments: measurement of phosphorylated and total proteins on the same bead
Gain valuable insights into the capabilities of the xMAP INTELLIFLEX DR-SE instrument and ProcartaPlex Signaling assays. This webinar is beneficial for scientists interested in advancing their research through the utilization of advanced technology and innovative assays. Thanks to our speakers, Sherry Dunbar and Sigrun Badrnya.
Take advantage of this resource and watch this webinar now.
Overview of cell signaling
Cell signaling is a complex process by which cells communicate with each other to coordinate their activities and respond to changes in their environment. This process involves transmission of signals between cells via various signaling pathways, which are typically initiated by the binding of a ligand to a receptor on the surface of the cell. There are several different types of signaling pathways that encompass diverse mechanisms for transmitting signals between cells.
Types of signaling pathways:
- Intracellular signaling pathways involve the transmission of signals within a single cell, typically through the activation of various protein kinases and other signaling molecules.
- Paracrine signaling pathways involve the transmission of signals between neighboring cells, while autocrine signaling pathways involve the transmission of signals from a cell to itself.
- Endocrine signaling pathways involve the transmission of signals throughout the body via the bloodstream.
Regardless of the type of signaling pathway involved, cell signaling enables cells to coordinate their activities and respond appropriately to changes in their environment. This process is critical for maintaining homeostasis within the body and ensures that cells can carry out their various functions in a coordinated and efficient manner.
One of the most extensively studied signaling pathways with significant relevance is the Akt pathway as it plays a critical role in regulating cell growth, survival, and metabolism. The Akt pathway is activated by various extracellular signals, including growth factors, hormones, and cytokines. Once activated, the Akt pathway regulates a wide range of cellular processes, including the regulation of glucose metabolism, protein synthesis, and cell proliferation. It does this by phosphorylating a variety of downstream targets, including the mTOR complex and glycogen synthase kinase 3 (GSK3) (Figure 1).
Learn more about cell signaling pathways
Figure 1. The Akt signaling pathway. The Akt signaling pathway is activated via growth factors such as insulin or Insulin Growth Factor (IGF) promoting phosphorylation of corresponding receptor tyrosine kinases (RTKs) like IGF-1R and propagating further signal transduction through phosphorylation of downstream targets such as IRS-1, Akt, GSK-3 beta, p70S6K, mTOR or CREB to regulate cell processes that are associated with tumor development, including regulators of apoptosis, gene transcription, cell cycle progression, and cellular metabolism.
General testing methods
One of the most common techniques to study cell signaling pathways is the use of biochemical and protein assays (Table 1). These assays involve the isolation and manipulation of cellular components to investigate the activation and regulation of specific signaling molecules within the pathway.
Examples of assays include:
- Immunoblotting and Western blotting (or Western Blot), which allows for the detection and quantification of specific proteins
- Immunoprecipitation, which can be used to study protein-protein interactions
- Enzyme activity assays, which measure the activity of enzymes involved in the signaling pathway
- ELISA (enzyme-linked immunosorbent assay) which measures the concentration of a specific protein
- Multiplex assays, like ProcartaPlex, which quantify multiple proteins simultaneously using bead-based technology
Table 1. Comparison table of some selected assays.
| Western blot | ELISA | ProcartaPlex | ProcartaPlex Dual Reporter |
|---|---|---|---|---|
| Best for | Validation, confirmation and visual identification of proteins | Single-target analysis with specificity and sensitivity | High-Throughput, profile more biomarker with less sample | Multiplex and measure two parameters of the same protein |
| Sample type | Various (e.g., lysate and purified proteins) | Various (cell and tissue lysates, plasma, serum, other body fluids, cell culture supernatants, etc.) | ||
| Protein Form | Denatured | Native | ||
| Protein extraction required | Yes | No | ||
| Quantification | Semi/relative quantitative | Quantitative; via calibration curve | ||
Detection range | Low to medium concentration | Low to high concentration | ||
| Protein size | Size separation and determination of protein MW | No information about the protein size | ||
Multiplexing | Up to 4-Plex fluorescent multiplexing, or Strip and reprobe chemiluminescent detection | No | Up to 80 analytes | Up to 2 parameters for 8 analytes |
ELISA and bead-based testing
ELISA and bead based multiplexing assays are widely used tools for studying cell signaling pathways. The main reason for the use of immunoassays are their sensitivity, specificity, simple protocol, and high throughput capabilities. Few examples of published data using Phospho-ELISA kits are listed in Table 2. In addition, both ELISA and ProcartaPlex assays demonstrate high correlation to other conventional laboratory methods like Western Blot. Through the utilization of ELISA and ProcartaPlex assays, researchers can elucidate the intricate mechanisms of cell signaling pathways, thereby facilitating the advancement of innovative therapeutic interventions aimed at addressing diverse diseases.
Table 2. List of publications highlighting immunological-based assays for studying cell signaling.
| Reference | Publication title | Target proteins/Panels used | Testing method |
|---|---|---|---|
| Vallés-Martí et al. 2023 | In-depth phosphoproteomics study for identification of hyperactive kinases in pancreatic ductal adenocarcinoma (PDAC). | EGFR | EGFR (Full-length) Human ELISA Kit |
| EGFR (Phospho) | EGFR (Phospho) [pY1173] Human ELISA Kit | ||
| Walker et al. 2023 | Identification and functional validation of Twinfilin-2 (TWF2) as a potential regulator of thin dendritic spine length. | Tau | Tau (Total) Human ELISA Kit |
| Tau (Phospho) | Tau (Phospho) [pS199] Human ELISA Kit | ||
| Tau (Phospho) [pT231] Human ELISA Kit | |||
| Tau (Phospho) [pS396] Human ELISA Kit | |||
| Scuruchi et al. 2023 | Endocan acts as a key molecule in inflamed articular chondrocytes that modulates proangiogenic factors expression and NF-kB activity. | NF-kB (Phospho) | NF-kB p65 (Phospho) [pS536] Human InstantOne ELISA Kit |
| Inostroza-Nieves et al. 2023 | Endothelin-1 (ET-1) promotes CREB activation and recruitment to MHC promoter in endothelial cells. | CREB | CREB (Total) Human ELISA Kit |
| CREB (Phospho) | |||
| Tamura et al. 2023 | Somatostatin receptor (SSTR5) antagonists may represent a novel and attractive therapeutic agent for type 2 diabetes. | AKT | AKT (Total) Human ELISA Kit |
| AKT (Phospho) | AKT (Phospho) [pS473] Human ELISA Kit | ||
| J et al. 2022 | Interleukin 8 signaling in intestinal epithelial cell line, HT-29 cells. | NF-kB | NF-κB p65 (Total) InstantOne ELISA Kit |
| p38, p38 (Phospho) | p38 (Total/Phospho) InstantOne ELISA Kit | ||
| ERK, ERK (Phospho) | ERK 1/2 (Total/Phospho) InstantOne ELISA Kit | ||
| Cornwell et al. 2022 | PI3k/Akt and NF-kB are critical for LPS-induced Glut1 expression. | NF-kB (Phospho) | NF-κB phospho-p65 InstantOne ELISA Kit |
| AKT (Phospho) | AKT (Phospho) [pS473] Multispecies InstantOne ELISA Kit | ||
| Wang et al. 2022 | Development of IL-2K35C-moFA and its potential as an immunotherapeutic agent for cancer therapy. | STAT5 (Phospho) | STAT5 alpha/beta (Phospho) [pY694/pY699] Human InstantOne ELISA Kit |
| Marfella et al. 2022 | Novel insights into pathophysiology of diabetic cardiomyopathy. | IRS1 (Phospho) | IRS1 (Phospho) [pS312] Human ELISA Kit |
| Oliveira-Santos et al. 2022 | Sunitinib could serve as a novel treatment for skeletal and cardiac muscle dysfunction in Duchenne muscular dystrophy (DMD). | STAT3 (Phospho) | STAT3 (Phospho) [pY705] Multispecies ELISA Kit |
| Okabe et al. 2022 | Administration of PFKFB3 and PFKFB4 inhibitors stops the growth of myeloma cells and enhances the cytotoxic effects of proteasome inhibitor, carfilzomib in hypoxic conditions. | NF-kB (Phospho) | NF-kB p65 (Phospho) [pS536] Human InstantOne ELISA Kit |
| p38 (Phospho) | p38 MAPK (Phospho) [pT180/pY182] Multispecies InstantOne ELISA Kit | ||
| Miyagishima et al. 2021 | Novel insights into the role of CTRP5 in the retinal pigment epithelium (RPE) and potential treatment strategies for late-onset retinal degeneration (L-ORD) patients. | AMPKα (Phospho) | AMPK alpha-1,2 (Phospho) [pT172] Human ELISA Kit |
| Nasca et al. 2021 | Exosomes might act as novel regulators of glutamate-related neuroplasticity in affective diseases, such as depression. | IRS1 (Phospho) |
Phospho-specific ELISA kits
Abnormal phosphorylation states are implicated in many human diseases, including cancer. Studying the phosphorylation and post-translational modification of key protein targets is essential for better understanding of the mechanisms behind the disease. Invitrogen ELISA kits specific for the phosphorylation states for several key targets and signaling pathways implicated in various diseases, including STAT3, CREB, PRAS40, p38, AMPK alpha, PTK2, and Caspase 3 signaling are listed in Table 3. These kits are available in two different formats–standard sandwich ELISA and InstantOne ELISA kits. Both types of kits are manufactured to help ensure excellent quality and reproducibility. The kits must meet quality-controlled specifications for sensitivity, dynamic range, precision, specificity, recovery, and lot-to-lot consistency.
Popular phosphorylated protein targets and ELISA performance data
Table 3. View our ELISA kits for the following popular targets:
Precoated Phospho-ELISA kits
Invitrogen precoated phospho-ELISA kits use a standard sandwich ELISA format involving pre-coated capture antibody (target-specific) to which the sample or standard and detection reagents are added sequentially. These kits come with protein standards to generate a standard curve for quantitative results and are designed to detect and quantify phosphorylated proteins in cell lysates. Figure 2 shows the level of total AKT and AKT [pS473] in Jurkat cells and demonstrates the relationship between AKT phosphorylation and PI-3 kinase activity. The specificity of precoated Phospho-ELISA kits has been confirmed by peptide competition assay (Figure 3) while the analytical sensitivity is determined by comparing western blot and ELISA against known quantities of phosphorylated proteins such as AKT [pS473] (Figure 4).
Benefits of precoated Phospho-ELISA kits:
- Quantitation—get quantitative data to complement western blot images
- Specificity—two antibodies directed against the analyte provide better specificity than western blot
- Sensitivity—more sensitive than western blot with typically only 2,000–3,000 cells needed
- Medium throughput—96-well format, results in 4 hours, no densitometry analysis needed
Figure 2. Measurement of Total vs. phosphorylated AKT in Jurkat cells using AKT ELISA. Jurkat cells were treated with increasing concentrations (0–500 nM) of wortmannin (a PI-3 kinase inhibitor). Cells were lysed and assayed in parallel for AKT [Total] and phosphorylated AKT [pS473] using AKT (Total) Human ELISA Kit and AKT (Phospho) [pS473] Human ELISA Kit ,respectively. The graph shows that the amount of total AKT remained comparable, however the levels of phosphorylation at serine 473 decreased in a dose-dependent manner with increase in wortmannin.
Figure 3. Specificity of AKT [pS473] ELISA. Peptide competition assay was conducted to confirm the specificity of AKT [pS473]. Titration of a specific blocking peptide to phosphorylation of AKT [pS473], was measured using AKT (Phospho) [pS473] Human ELISA Kit. The graph shows that only the peptide corresponding to the region surrounding serine 473 blocks the ELISA signal.
Figure 4. Analytical sensitivity of AKT [pS473] ELISA. Jurkat cells were cultured under optimal conditions and phosphorylated AKT cell extracts were obtained. Stimulation of phosphorylated AKT [pS473] was measured in cell lysate using AKT (Phospho) [pS473] Human ELISA Kit and Western blot. The analytical sensitivity of AKT [pS473] ELISA was determined by adding two standard deviations to the mean O.D. obtained from 30 assays of the zero standard. The value of <0.8 Units/mL corresponds to the amount of AKT [pS473] extracted from 4000 Jurkat cells. Results showed that the sensitivity of the ELISA is ~2-fold greater than that of western blot when tested against known quantities of AKT [pS473].
InstantOne Phospho-ELISA kits
Invitrogen InstantOne Phospho-ELISA kits come with all reagents used in a traditional sandwich ELISA that are added in solution to an InstantOne assay plate. These kits have a condensed workflow wherein the target analyte binds to two sandwich ELISA antibodies in solution while the capture antibody binds to the plate through a proprietary mechanism. These assay plates offer fast analysis of samples and enable a simple one-wash protocol. Detection of the specific analyte is performed with 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric substrate.
These kits are engineered for accurate measurement of total and/or phosphorylated proteins in cell lysates. InstantOne assays have been validated by ELISA for the detection of several key targets including NF-kB, p38 MAPK (Figure 5), SMAD1 (Figure 6) and evaluated for specificity by immunoblot analysis.
Benefits of InstantOne Phospho-ELISA kits:
- Flexibility—enables the detection of total and/or phospho protein levels of a single protein on a single plate
- Specificity—two antibodies directed against the analyte provide better specificity than western blot
- Sensitivity—more sensitive than western blot with typically only 2,000-3,000 cells needed
- High throughput—1 hour and 1 wash protocol
Figure 5. Detection of human p38 MAPK [pT180/pY182] in HEK293 cells using InstantOne ELISA. HEK293 cells were treated with 2 µg/mL of anisomycin (a potent activator of p38/JNK MAPK pathway) for 30 min. Untreated HEK293 cells (-) served as negative control. These cells were analyzed by Western Blot (right) and ELISA (left) using p38 MAPK (Phospho) [pT180/pY182] Multispecies InstantOne ELISA Kit. The intensity of the band at 38 kDa as detected by immunoblot correlates with InstantOne ELISA absorption values.
Figure 6. Detection of SMAD1 (Total/Phospho) in C2C12 cells using InstantOne ELISA. C2C12 cells were either inhibited with dosomorphin (50 mM) for 60 min) (-) or stimulated with BMP4 (10 ng/mL) for 30 min (+). Lysates were prepared as described in the InstantOne ELISA user manual. Cells (10 µg/assay) were analyzed for total SMAD1 (left) or phosphorylated SMAD1 [S463/S465] (right) using SMAD1 (Total/Phospho) InstantOne ELISA Kit.
Cell signaling ProcartaPlex multiplex immunoassays
ProcartaPlex is a Luminex bead-based multiplex immunoassay that enables simultaneous quantitation of multiple analytes within a single well. Total and/or phosphorylated forms of proteins can be quantified simultaneously. This helps to unveil the complex interactions in studying cell signaling pathways.
Select from our classic ProcartaPlex formats that are specifically designed to measure total or phosphorylated proteins or discover the novel ProcartaPlex Dual Reporter assays for use on the Luminex xMAP INTELLIFLEX DR-SE instrument that offers the unique capability to quantify both the phosphorylated and total form of up to 8 proteins at once on the same bead (Table 4).
Benefits of ProcartaPlex cell signaling assays:
- Comparability—obtain comparable results to ELISA (Figure 7) and Western Blotanalysis (Figure 8) with the added value of multiplexing
- Broad dynamic range and enhanced sensitivity—enabling the detection of even low-abundance proteins and signaling molecules (Figure 9)
- Dual Reporter multiplexing—allowing simultaneous detection of multiple phosphorylated and total proteins on the same bead in a single well (Figure 10)
This multiplexing capability saves time and valuable sample material, helping to obtain comprehensive data more efficiently. With less hands-on time and increased throughput compared to ELISA and Western Blot, multiplexing assays significantly enhance productivity in signaling research.
Table 4. ProcartaPlex cell signaling assays are available in multiple formats.
| ProcartaPlex | ProcartaPlex Dual Reporter |
| Total protein (only) |  | x |
| Phosphorylated protein (only) | | x |
Total and phosphorylated proteins* | x | |
| Multiplex capacity for cell signaling | Up to 8 | Up to 16 (2 parameters of 8 analytes) |
| Plate Format | 96-well | 96-well |
| Luminex instrument | xMAP INTELLIFLEX and xMAP INTELLIFLEX DR-SE System FLEXMAP 3D System Luminex 200 System | Only xMAP INTELLIFLEX DR-SE System |
| Sample volume | 25 µL of prediluted lysates | 25 µL of prediluted lysates |
| Read time | 4 hours | 4 hours |
| Assay format | ||
| Preconfigured Panels | | |
| Mix & Match | | |
| Simplex kits | | x |
Learn more about ProcartaPlex multiplex immunoassays
How it works:
A) For ProcartaPlex assays the Luminex beads are coupled with capture antibodies that specifically bind to signaling proteins, regardless of their phosphorylation state. The conjugation of a specific antibody to a distinct bead number enables the analysis of multiple analytes in a single well. Separate panels are created for multiplex analysis of total or phosphorylated signaling proteins using non-phospho-specific or phospho-specific detection antibodies with PE-dye for reporter channel 1 (532nm), making them compatible with all Luminex instruments.
(B) For ProcartaPlex Dual Reporter assays the same capture antibodies are applied, but for quantitation of phosphorylated signaling proteins a phospho-specific detection antibody with BV421-conjugation is used that leverages the additional reporter channel 2 (405 nm) of the Luminex xMAP INTELLIFLEX DR-SE instrument. Total proteins are detected through reporter channel 1 (532 nm) with the PE dye. This approach enables the simultaneous quantitation of both total and phosphorylated proteins in a single well on the same bead.
Benefits of multiplexing cell signaling pathways
Western blotting is a commonly used technique for identifying and detecting protein targets and their post-translational modification states. The simplicity and flexibility of the western blotting technique is especially useful for foundational work and validation. However, in cell signaling research, the need to analyze multiple proteins and their phosphorylation status is often combined with a high number of samples. This fact makes multiplexing a valuable tool in this area of research as it allows to simultaneously quantify multiple analytes in a single sample while saving time, sample volume and enabling high throughput. The multiplexing protocol is like an ELISA assay. ProcartaPlex assays have demonstrated a high correlation with ELISA (Figure 7) and Western Blot (Figure 8) and showed excellent sensitivity (Figure 9). In addition, the novel ProcartaPlex Dual Reporter Assays allows simultaneous quantification of total and phosphorylated proteins on the same bead in a single well (Figure 10).
Preconfigured signaling multiplex immunoassay panels and performance data
Figure 7. Comparison data to demonstrate correlation of ProcartaPlex signaling assays with conventional ELISA. Lysates of various cell lines were used in duplicates and run in parallel on ProcartaPlex Signaling Simplex kits, Akt [pS473] Human ProcartaPlex Simplex Kit, PRAS40 [pT246] Human ProcartaPlex Simplex Kit and CREB [pS133] Human ProcartaPlex Simplex Kit and ELISA kits, AKT (Phospho) [pS473] Human ELISA Kit , PRAS40 (Phospho) [pT246] Human ELISA Kit and CREB (Phospho) [pS133] Human ELISA Kit. Presented data of the measured concentrations of Akt [pS473], PRAS40 [pT246] and CREB [pS133] show high correlation (R2 > 0.8). This demonstrates that ProcartaPlex Signaling Simplex kits achieve quantitative results comparable to traditional ELISA kits with the additional benefits of multiplexing including reduced sample volume, shorter assay/hands-on time and lower costs per data point.
Figure 8. Comparison data for ProcartaPlex and western blot. Performance comparison of protein analysis using western blot and ProcartaPlex Human Akt Pathway Dual Reporter Panel, 2x8plex demonstrates a high correlation between the two assay systems. Western blot data shows PRAS40 [pT246] levels (40 kDa) in lysed NIH-3T3 cells. Equal amounts of protein (10 µg) were used for western blot and ProcartaPlex assays. ProcartaPlex assay was performed according to the manufacturers’ instructions. For western blot analysis, protein samples were electrophoresed using Bolt Bis-Tris Plus Mini Protein Gels, 4-12%, 1.0 mm, WedgeWell format, XCell SureLock Mini-Cell and XCell II Blot Module and PageRuler Plus Prestained Protein Ladder, 10 to 250 kDa. Resolved proteins were then transferred onto an iBlot 2 Transfer Stacks, nitrocellulose, mini with iBlot 2 Dry Blotting System. The membrane was probed with the relevant primary and secondary antibody after blocking using iBind Solution Kit. Chemiluminescent detection was performed using SuperSignal West Pico PLUS Chemiluminescent Substrate.
Figure 9. Comparison of analytical sensitivity of ProcartaPlex Signaling Assay and western blot. Jurkat cells were cultured under optimal growing conditions and cell lysate was used to measure phosphorylated CREB. Same amount of protein (10 µg) was used for a serial dilution 1:2 and measured with CREB [pS133] Human ProcartaPlex Simplex Kit and western blot assay. For western blot, the same phospho-specific detection antibody against PRAS40 [pT246] was used as included in the Simplex kit. The lowest concentration measured with western blot was 5 µg/well while the lowest concentration measured with ProcartaPlex Simplex kit was 0.6 µg/well.
Figure 10. Correlation between conventional ProcartaPlex and ProcartaPlex Dual Reporter format.ProcartaPlex Human Akt Pathway Dual Reporter Panel, 2x8plex was utilized alongside the ProcartaPlex Human Akt Pathway Panel, total, 8plex and ProcartaPlex Human Akt Pathway Panel, phospho, 8plex to assess total or phosphorylated proteins involved in the Akt signaling pathway. The obtained data demonstrate a strong correlation between the Dual Reporter Panel and the conventional panels for either total or phosphorylated proteins (R2 > 0.9).
Table 5. Preconfigured ProcartaPlex multiplex immunoassay panels for studying cell signaling.
| Dual Reporter panels for quantitation of total and phosphorylated proteins | ||
| Product name | Size | Cat. No. |
ProcartaPlex Human Akt Pathway Dual Reporter Panel, 2x 8plex Target list [bead region]: Akt + Akt[pS473] [47], CREB + CREB[pS133] [43], GSK-3 beta + GSK-3 beta[pS9] [56], IGF-1R + IGF-1R[pYpY1135/1136] [34], IRS-1 + IRS-1[pS312] [37], mTor + mTOR[Ser2448] [26], PRAS40 + PRAS40[pT246] [42], p70S6K + p70S6K[pTpS421/424] [27] | 96 well | EPX080-97200-DR |
See our latest product releases: Singleplex and Multiplex Bead Based Immunoassays
| Panels for quantitation of total proteins | ||
| Product name | Size | Cat. No. |
ProcartaPlex Human Akt Pathway Panel (total), 8plex Akt [47], CREB [43], GSK-3 beta [56], IGF-1R [34], IRS-1 [37], mTor [26], PRAS40 [42], p70S6K [27] | 96 well | EPX080-97000-901 |
| Panels for quantitation of phosphorylated protein | ||
| Product name | Size | Cat. No. |
ProcartaPlex Human Akt Pathway Panel (phospho), 8plex Target list [bead region]: Akt[pS473] [47], CREB[PS133] [43], GSK-3 beta[pS9] [56], IGF-1R[pYpY1135/1136] [34], IRS-1[pS312] [37], mTOR[Ser2448] [26], PRAS40[pT246] [42], p70S6K[pTpS421/424] [27] | 96 well | EPX080-97100-PHO |
Please note: The ProcartaPlex Simplex kits for total proteins can be combined with one another. The same is true for Simplex kits for phosphorylated proteins. Combining Simplex kits for total and phosphorylated proteins is not possible. If you wish to quantify both total and phosphorylated proteins in a single panel, you can use the ProcartaPlex Panel Configurator to design your panel. However, it is important to note that these panels can only be run on the Luminex xMAP INTELLIFLEX DR-SE instrument.
Learn more about Luminex instrument
Overview of cell signaling
Cell signaling is a complex process by which cells communicate with each other to coordinate their activities and respond to changes in their environment. This process involves transmission of signals between cells via various signaling pathways, which are typically initiated by the binding of a ligand to a receptor on the surface of the cell. There are several different types of signaling pathways that encompass diverse mechanisms for transmitting signals between cells.
Types of signaling pathways:
- Intracellular signaling pathways involve the transmission of signals within a single cell, typically through the activation of various protein kinases and other signaling molecules.
- Paracrine signaling pathways involve the transmission of signals between neighboring cells, while autocrine signaling pathways involve the transmission of signals from a cell to itself.
- Endocrine signaling pathways involve the transmission of signals throughout the body via the bloodstream.
Regardless of the type of signaling pathway involved, cell signaling enables cells to coordinate their activities and respond appropriately to changes in their environment. This process is critical for maintaining homeostasis within the body and ensures that cells can carry out their various functions in a coordinated and efficient manner.
One of the most extensively studied signaling pathways with significant relevance is the Akt pathway as it plays a critical role in regulating cell growth, survival, and metabolism. The Akt pathway is activated by various extracellular signals, including growth factors, hormones, and cytokines. Once activated, the Akt pathway regulates a wide range of cellular processes, including the regulation of glucose metabolism, protein synthesis, and cell proliferation. It does this by phosphorylating a variety of downstream targets, including the mTOR complex and glycogen synthase kinase 3 (GSK3) (Figure 1).
Learn more about cell signaling pathways
Figure 1. The Akt signaling pathway. The Akt signaling pathway is activated via growth factors such as insulin or Insulin Growth Factor (IGF) promoting phosphorylation of corresponding receptor tyrosine kinases (RTKs) like IGF-1R and propagating further signal transduction through phosphorylation of downstream targets such as IRS-1, Akt, GSK-3 beta, p70S6K, mTOR or CREB to regulate cell processes that are associated with tumor development, including regulators of apoptosis, gene transcription, cell cycle progression, and cellular metabolism.
General testing methods
One of the most common techniques to study cell signaling pathways is the use of biochemical and protein assays (Table 1). These assays involve the isolation and manipulation of cellular components to investigate the activation and regulation of specific signaling molecules within the pathway.
Examples of assays include:
- Immunoblotting and Western blotting (or Western Blot), which allows for the detection and quantification of specific proteins
- Immunoprecipitation, which can be used to study protein-protein interactions
- Enzyme activity assays, which measure the activity of enzymes involved in the signaling pathway
- ELISA (enzyme-linked immunosorbent assay) which measures the concentration of a specific protein
- Multiplex assays, like ProcartaPlex, which quantify multiple proteins simultaneously using bead-based technology
Table 1. Comparison table of some selected assays.
| Western blot | ELISA | ProcartaPlex | ProcartaPlex Dual Reporter |
|---|---|---|---|---|
| Best for | Validation, confirmation and visual identification of proteins | Single-target analysis with specificity and sensitivity | High-Throughput, profile more biomarker with less sample | Multiplex and measure two parameters of the same protein |
| Sample type | Various (e.g., lysate and purified proteins) | Various (cell and tissue lysates, plasma, serum, other body fluids, cell culture supernatants, etc.) | ||
| Protein Form | Denatured | Native | ||
| Protein extraction required | Yes | No | ||
| Quantification | Semi/relative quantitative | Quantitative; via calibration curve | ||
Detection range | Low to medium concentration | Low to high concentration | ||
| Protein size | Size separation and determination of protein MW | No information about the protein size | ||
Multiplexing | Up to 4-Plex fluorescent multiplexing, or Strip and reprobe chemiluminescent detection | No | Up to 80 analytes | Up to 2 parameters for 8 analytes |
ELISA and bead-based testing
ELISA and bead based multiplexing assays are widely used tools for studying cell signaling pathways. The main reason for the use of immunoassays are their sensitivity, specificity, simple protocol, and high throughput capabilities. Few examples of published data using Phospho-ELISA kits are listed in Table 2. In addition, both ELISA and ProcartaPlex assays demonstrate high correlation to other conventional laboratory methods like Western Blot. Through the utilization of ELISA and ProcartaPlex assays, researchers can elucidate the intricate mechanisms of cell signaling pathways, thereby facilitating the advancement of innovative therapeutic interventions aimed at addressing diverse diseases.
Table 2. List of publications highlighting immunological-based assays for studying cell signaling.
| Reference | Publication title | Target proteins/Panels used | Testing method |
|---|---|---|---|
| Vallés-Martí et al. 2023 | In-depth phosphoproteomics study for identification of hyperactive kinases in pancreatic ductal adenocarcinoma (PDAC). | EGFR | EGFR (Full-length) Human ELISA Kit |
| EGFR (Phospho) | EGFR (Phospho) [pY1173] Human ELISA Kit | ||
| Walker et al. 2023 | Identification and functional validation of Twinfilin-2 (TWF2) as a potential regulator of thin dendritic spine length. | Tau | Tau (Total) Human ELISA Kit |
| Tau (Phospho) | Tau (Phospho) [pS199] Human ELISA Kit | ||
| Tau (Phospho) [pT231] Human ELISA Kit | |||
| Tau (Phospho) [pS396] Human ELISA Kit | |||
| Scuruchi et al. 2023 | Endocan acts as a key molecule in inflamed articular chondrocytes that modulates proangiogenic factors expression and NF-kB activity. | NF-kB (Phospho) | NF-kB p65 (Phospho) [pS536] Human InstantOne ELISA Kit |
| Inostroza-Nieves et al. 2023 | Endothelin-1 (ET-1) promotes CREB activation and recruitment to MHC promoter in endothelial cells. | CREB | CREB (Total) Human ELISA Kit |
| CREB (Phospho) | |||
| Tamura et al. 2023 | Somatostatin receptor (SSTR5) antagonists may represent a novel and attractive therapeutic agent for type 2 diabetes. | AKT | AKT (Total) Human ELISA Kit |
| AKT (Phospho) | AKT (Phospho) [pS473] Human ELISA Kit | ||
| J et al. 2022 | Interleukin 8 signaling in intestinal epithelial cell line, HT-29 cells. | NF-kB | NF-κB p65 (Total) InstantOne ELISA Kit |
| p38, p38 (Phospho) | p38 (Total/Phospho) InstantOne ELISA Kit | ||
| ERK, ERK (Phospho) | ERK 1/2 (Total/Phospho) InstantOne ELISA Kit | ||
| Cornwell et al. 2022 | PI3k/Akt and NF-kB are critical for LPS-induced Glut1 expression. | NF-kB (Phospho) | NF-κB phospho-p65 InstantOne ELISA Kit |
| AKT (Phospho) | AKT (Phospho) [pS473] Multispecies InstantOne ELISA Kit | ||
| Wang et al. 2022 | Development of IL-2K35C-moFA and its potential as an immunotherapeutic agent for cancer therapy. | STAT5 (Phospho) | STAT5 alpha/beta (Phospho) [pY694/pY699] Human InstantOne ELISA Kit |
| Marfella et al. 2022 | Novel insights into pathophysiology of diabetic cardiomyopathy. | IRS1 (Phospho) | IRS1 (Phospho) [pS312] Human ELISA Kit |
| Oliveira-Santos et al. 2022 | Sunitinib could serve as a novel treatment for skeletal and cardiac muscle dysfunction in Duchenne muscular dystrophy (DMD). | STAT3 (Phospho) | STAT3 (Phospho) [pY705] Multispecies ELISA Kit |
| Okabe et al. 2022 | Administration of PFKFB3 and PFKFB4 inhibitors stops the growth of myeloma cells and enhances the cytotoxic effects of proteasome inhibitor, carfilzomib in hypoxic conditions. | NF-kB (Phospho) | NF-kB p65 (Phospho) [pS536] Human InstantOne ELISA Kit |
| p38 (Phospho) | p38 MAPK (Phospho) [pT180/pY182] Multispecies InstantOne ELISA Kit | ||
| Miyagishima et al. 2021 | Novel insights into the role of CTRP5 in the retinal pigment epithelium (RPE) and potential treatment strategies for late-onset retinal degeneration (L-ORD) patients. | AMPKα (Phospho) | AMPK alpha-1,2 (Phospho) [pT172] Human ELISA Kit |
| Nasca et al. 2021 | Exosomes might act as novel regulators of glutamate-related neuroplasticity in affective diseases, such as depression. | IRS1 (Phospho) |
Phospho-specific ELISA kits
Abnormal phosphorylation states are implicated in many human diseases, including cancer. Studying the phosphorylation and post-translational modification of key protein targets is essential for better understanding of the mechanisms behind the disease. Invitrogen ELISA kits specific for the phosphorylation states for several key targets and signaling pathways implicated in various diseases, including STAT3, CREB, PRAS40, p38, AMPK alpha, PTK2, and Caspase 3 signaling are listed in Table 3. These kits are available in two different formats–standard sandwich ELISA and InstantOne ELISA kits. Both types of kits are manufactured to help ensure excellent quality and reproducibility. The kits must meet quality-controlled specifications for sensitivity, dynamic range, precision, specificity, recovery, and lot-to-lot consistency.
Popular phosphorylated protein targets and ELISA performance data
Table 3. View our ELISA kits for the following popular targets:
Precoated Phospho-ELISA kits
Invitrogen precoated phospho-ELISA kits use a standard sandwich ELISA format involving pre-coated capture antibody (target-specific) to which the sample or standard and detection reagents are added sequentially. These kits come with protein standards to generate a standard curve for quantitative results and are designed to detect and quantify phosphorylated proteins in cell lysates. Figure 2 shows the level of total AKT and AKT [pS473] in Jurkat cells and demonstrates the relationship between AKT phosphorylation and PI-3 kinase activity. The specificity of precoated Phospho-ELISA kits has been confirmed by peptide competition assay (Figure 3) while the analytical sensitivity is determined by comparing western blot and ELISA against known quantities of phosphorylated proteins such as AKT [pS473] (Figure 4).
Benefits of precoated Phospho-ELISA kits:
- Quantitation—get quantitative data to complement western blot images
- Specificity—two antibodies directed against the analyte provide better specificity than western blot
- Sensitivity—more sensitive than western blot with typically only 2,000–3,000 cells needed
- Medium throughput—96-well format, results in 4 hours, no densitometry analysis needed
Figure 2. Measurement of Total vs. phosphorylated AKT in Jurkat cells using AKT ELISA. Jurkat cells were treated with increasing concentrations (0–500 nM) of wortmannin (a PI-3 kinase inhibitor). Cells were lysed and assayed in parallel for AKT [Total] and phosphorylated AKT [pS473] using AKT (Total) Human ELISA Kit and AKT (Phospho) [pS473] Human ELISA Kit ,respectively. The graph shows that the amount of total AKT remained comparable, however the levels of phosphorylation at serine 473 decreased in a dose-dependent manner with increase in wortmannin.
Figure 3. Specificity of AKT [pS473] ELISA. Peptide competition assay was conducted to confirm the specificity of AKT [pS473]. Titration of a specific blocking peptide to phosphorylation of AKT [pS473], was measured using AKT (Phospho) [pS473] Human ELISA Kit. The graph shows that only the peptide corresponding to the region surrounding serine 473 blocks the ELISA signal.
Figure 4. Analytical sensitivity of AKT [pS473] ELISA. Jurkat cells were cultured under optimal conditions and phosphorylated AKT cell extracts were obtained. Stimulation of phosphorylated AKT [pS473] was measured in cell lysate using AKT (Phospho) [pS473] Human ELISA Kit and Western blot. The analytical sensitivity of AKT [pS473] ELISA was determined by adding two standard deviations to the mean O.D. obtained from 30 assays of the zero standard. The value of <0.8 Units/mL corresponds to the amount of AKT [pS473] extracted from 4000 Jurkat cells. Results showed that the sensitivity of the ELISA is ~2-fold greater than that of western blot when tested against known quantities of AKT [pS473].
InstantOne Phospho-ELISA kits
Invitrogen InstantOne Phospho-ELISA kits come with all reagents used in a traditional sandwich ELISA that are added in solution to an InstantOne assay plate. These kits have a condensed workflow wherein the target analyte binds to two sandwich ELISA antibodies in solution while the capture antibody binds to the plate through a proprietary mechanism. These assay plates offer fast analysis of samples and enable a simple one-wash protocol. Detection of the specific analyte is performed with 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric substrate.
These kits are engineered for accurate measurement of total and/or phosphorylated proteins in cell lysates. InstantOne assays have been validated by ELISA for the detection of several key targets including NF-kB, p38 MAPK (Figure 5), SMAD1 (Figure 6) and evaluated for specificity by immunoblot analysis.
Benefits of InstantOne Phospho-ELISA kits:
- Flexibility—enables the detection of total and/or phospho protein levels of a single protein on a single plate
- Specificity—two antibodies directed against the analyte provide better specificity than western blot
- Sensitivity—more sensitive than western blot with typically only 2,000-3,000 cells needed
- High throughput—1 hour and 1 wash protocol
Figure 5. Detection of human p38 MAPK [pT180/pY182] in HEK293 cells using InstantOne ELISA. HEK293 cells were treated with 2 µg/mL of anisomycin (a potent activator of p38/JNK MAPK pathway) for 30 min. Untreated HEK293 cells (-) served as negative control. These cells were analyzed by Western Blot (right) and ELISA (left) using p38 MAPK (Phospho) [pT180/pY182] Multispecies InstantOne ELISA Kit. The intensity of the band at 38 kDa as detected by immunoblot correlates with InstantOne ELISA absorption values.
Figure 6. Detection of SMAD1 (Total/Phospho) in C2C12 cells using InstantOne ELISA. C2C12 cells were either inhibited with dosomorphin (50 mM) for 60 min) (-) or stimulated with BMP4 (10 ng/mL) for 30 min (+). Lysates were prepared as described in the InstantOne ELISA user manual. Cells (10 µg/assay) were analyzed for total SMAD1 (left) or phosphorylated SMAD1 [S463/S465] (right) using SMAD1 (Total/Phospho) InstantOne ELISA Kit.
Cell signaling ProcartaPlex multiplex immunoassays
ProcartaPlex is a Luminex bead-based multiplex immunoassay that enables simultaneous quantitation of multiple analytes within a single well. Total and/or phosphorylated forms of proteins can be quantified simultaneously. This helps to unveil the complex interactions in studying cell signaling pathways.
Select from our classic ProcartaPlex formats that are specifically designed to measure total or phosphorylated proteins or discover the novel ProcartaPlex Dual Reporter assays for use on the Luminex xMAP INTELLIFLEX DR-SE instrument that offers the unique capability to quantify both the phosphorylated and total form of up to 8 proteins at once on the same bead (Table 4).
Benefits of ProcartaPlex cell signaling assays:
- Comparability—obtain comparable results to ELISA (Figure 7) and Western Blotanalysis (Figure 8) with the added value of multiplexing
- Broad dynamic range and enhanced sensitivity—enabling the detection of even low-abundance proteins and signaling molecules (Figure 9)
- Dual Reporter multiplexing—allowing simultaneous detection of multiple phosphorylated and total proteins on the same bead in a single well (Figure 10)
This multiplexing capability saves time and valuable sample material, helping to obtain comprehensive data more efficiently. With less hands-on time and increased throughput compared to ELISA and Western Blot, multiplexing assays significantly enhance productivity in signaling research.
Table 4. ProcartaPlex cell signaling assays are available in multiple formats.
| ProcartaPlex | ProcartaPlex Dual Reporter |
| Total protein (only) | | x |
| Phosphorylated protein (only) | | x |
Total and phosphorylated proteins* | x | |
| Multiplex capacity for cell signaling | Up to 8 | Up to 16 (2 parameters of 8 analytes) |
| Plate Format | 96-well | 96-well |
| Luminex instrument | xMAP INTELLIFLEX and xMAP INTELLIFLEX DR-SE System FLEXMAP 3D System Luminex 200 System | Only xMAP INTELLIFLEX DR-SE System |
| Sample volume | 25 µL of prediluted lysates | 25 µL of prediluted lysates |
| Read time | 4 hours | 4 hours |
| Assay format | ||
| Preconfigured Panels | | |
| Mix & Match | | |
| Simplex kits | | x |
Learn more about ProcartaPlex multiplex immunoassays
How it works:
A) For ProcartaPlex assays the Luminex beads are coupled with capture antibodies that specifically bind to signaling proteins, regardless of their phosphorylation state. The conjugation of a specific antibody to a distinct bead number enables the analysis of multiple analytes in a single well. Separate panels are created for multiplex analysis of total or phosphorylated signaling proteins using non-phospho-specific or phospho-specific detection antibodies with PE-dye for reporter channel 1 (532nm), making them compatible with all Luminex instruments.
(B) For ProcartaPlex Dual Reporter assays the same capture antibodies are applied, but for quantitation of phosphorylated signaling proteins a phospho-specific detection antibody with BV421-conjugation is used that leverages the additional reporter channel 2 (405 nm) of the Luminex xMAP INTELLIFLEX DR-SE instrument. Total proteins are detected through reporter channel 1 (532 nm) with the PE dye. This approach enables the simultaneous quantitation of both total and phosphorylated proteins in a single well on the same bead.
Benefits of multiplexing cell signaling pathways
Western blotting is a commonly used technique for identifying and detecting protein targets and their post-translational modification states. The simplicity and flexibility of the western blotting technique is especially useful for foundational work and validation. However, in cell signaling research, the need to analyze multiple proteins and their phosphorylation status is often combined with a high number of samples. This fact makes multiplexing a valuable tool in this area of research as it allows to simultaneously quantify multiple analytes in a single sample while saving time, sample volume and enabling high throughput. The multiplexing protocol is like an ELISA assay. ProcartaPlex assays have demonstrated a high correlation with ELISA (Figure 7) and Western Blot (Figure 8) and showed excellent sensitivity (Figure 9). In addition, the novel ProcartaPlex Dual Reporter Assays allows simultaneous quantification of total and phosphorylated proteins on the same bead in a single well (Figure 10).
Preconfigured signaling multiplex immunoassay panels and performance data
Figure 7. Comparison data to demonstrate correlation of ProcartaPlex signaling assays with conventional ELISA. Lysates of various cell lines were used in duplicates and run in parallel on ProcartaPlex Signaling Simplex kits, Akt [pS473] Human ProcartaPlex Simplex Kit, PRAS40 [pT246] Human ProcartaPlex Simplex Kit and CREB [pS133] Human ProcartaPlex Simplex Kit and ELISA kits, AKT (Phospho) [pS473] Human ELISA Kit , PRAS40 (Phospho) [pT246] Human ELISA Kit and CREB (Phospho) [pS133] Human ELISA Kit. Presented data of the measured concentrations of Akt [pS473], PRAS40 [pT246] and CREB [pS133] show high correlation (R2 > 0.8). This demonstrates that ProcartaPlex Signaling Simplex kits achieve quantitative results comparable to traditional ELISA kits with the additional benefits of multiplexing including reduced sample volume, shorter assay/hands-on time and lower costs per data point.
Figure 8. Comparison data for ProcartaPlex and western blot. Performance comparison of protein analysis using western blot and ProcartaPlex Human Akt Pathway Dual Reporter Panel, 2x8plex demonstrates a high correlation between the two assay systems. Western blot data shows PRAS40 [pT246] levels (40 kDa) in lysed NIH-3T3 cells. Equal amounts of protein (10 µg) were used for western blot and ProcartaPlex assays. ProcartaPlex assay was performed according to the manufacturers’ instructions. For western blot analysis, protein samples were electrophoresed using Bolt Bis-Tris Plus Mini Protein Gels, 4-12%, 1.0 mm, WedgeWell format, XCell SureLock Mini-Cell and XCell II Blot Module and PageRuler Plus Prestained Protein Ladder, 10 to 250 kDa. Resolved proteins were then transferred onto an iBlot 2 Transfer Stacks, nitrocellulose, mini with iBlot 2 Dry Blotting System. The membrane was probed with the relevant primary and secondary antibody after blocking using iBind Solution Kit. Chemiluminescent detection was performed using SuperSignal West Pico PLUS Chemiluminescent Substrate.
Figure 9. Comparison of analytical sensitivity of ProcartaPlex Signaling Assay and western blot. Jurkat cells were cultured under optimal growing conditions and cell lysate was used to measure phosphorylated CREB. Same amount of protein (10 µg) was used for a serial dilution 1:2 and measured with CREB [pS133] Human ProcartaPlex Simplex Kit and western blot assay. For western blot, the same phospho-specific detection antibody against PRAS40 [pT246] was used as included in the Simplex kit. The lowest concentration measured with western blot was 5 µg/well while the lowest concentration measured with ProcartaPlex Simplex kit was 0.6 µg/well.
Figure 10. Correlation between conventional ProcartaPlex and ProcartaPlex Dual Reporter format.ProcartaPlex Human Akt Pathway Dual Reporter Panel, 2x8plex was utilized alongside the ProcartaPlex Human Akt Pathway Panel, total, 8plex and ProcartaPlex Human Akt Pathway Panel, phospho, 8plex to assess total or phosphorylated proteins involved in the Akt signaling pathway. The obtained data demonstrate a strong correlation between the Dual Reporter Panel and the conventional panels for either total or phosphorylated proteins (R2 > 0.9).
Table 5. Preconfigured ProcartaPlex multiplex immunoassay panels for studying cell signaling.
| Dual Reporter panels for quantitation of total and phosphorylated proteins | ||
| Product name | Size | Cat. No. |
ProcartaPlex Human Akt Pathway Dual Reporter Panel, 2x 8plex Target list [bead region]: Akt + Akt[pS473] [47], CREB + CREB[pS133] [43], GSK-3 beta + GSK-3 beta[pS9] [56], IGF-1R + IGF-1R[pYpY1135/1136] [34], IRS-1 + IRS-1[pS312] [37], mTor + mTOR[Ser2448] [26], PRAS40 + PRAS40[pT246] [42], p70S6K + p70S6K[pTpS421/424] [27] | 96 well | EPX080-97200-DR |
See our latest product releases: Singleplex and Multiplex Bead Based Immunoassays
| Panels for quantitation of total proteins | ||
| Product name | Size | Cat. No. |
ProcartaPlex Human Akt Pathway Panel (total), 8plex Akt [47], CREB [43], GSK-3 beta [56], IGF-1R [34], IRS-1 [37], mTor [26], PRAS40 [42], p70S6K [27] | 96 well | EPX080-97000-901 |
| Panels for quantitation of phosphorylated protein | ||
| Product name | Size | Cat. No. |
ProcartaPlex Human Akt Pathway Panel (phospho), 8plex Target list [bead region]: Akt[pS473] [47], CREB[PS133] [43], GSK-3 beta[pS9] [56], IGF-1R[pYpY1135/1136] [34], IRS-1[pS312] [37], mTOR[Ser2448] [26], PRAS40[pT246] [42], p70S6K[pTpS421/424] [27] | 96 well | EPX080-97100-PHO |
Please note: The ProcartaPlex Simplex kits for total proteins can be combined with one another. The same is true for Simplex kits for phosphorylated proteins. Combining Simplex kits for total and phosphorylated proteins is not possible. If you wish to quantify both total and phosphorylated proteins in a single panel, you can use the ProcartaPlex Panel Configurator to design your panel. However, it is important to note that these panels can only be run on the Luminex xMAP INTELLIFLEX DR-SE instrument.
Learn more about Luminex instrument
Additional resources for cell signaling immunoassays
Related instruments
References
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For Research Use Only. Not for use in diagnostic procedures.