Cell Signaling ELISA Kits and Multiplex Immunoassays

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.

Search 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

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

(TOP)

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.

ProcartaPlex panel configurator

Learn more about ProcartaPlex multiplex immunoassays

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).

(TOP)

See our latest product releases: Singleplex and Multiplex Bead Based Immunoassays

(TOP)