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. We have ELISA kits specific for the phosphorylation states for many of the key targets and signaling pathways implicated in various diseases, including STAT3, CREB, PRAS40, p38, AMPK alpha, PTK2, and Caspase 3 signaling. These kits are available in two different formats–standard sandwich ELISA and InstantOne ELISA kits. Both type 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.

Explore 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 1 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 2) while the analytical sensitivity is determined by comparing western blot and ELISA against known quantities of phosphorylated proteins such as AKT [pS473] (Figure 3). Few examples of published data using precoated Phospho-ELISA kits have been highlighted in Table 1.

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 blotting
• Sensitivity—more sensitive than western blotting with only 2,000–3,000 cells typically needed
• Medium throughput—96-well format, results in 4 hours, no densitometry analysis needed

Precoated Phospho-ELISA kit contents:

Explore Precoated Phospho-ELISA kits

Figure 1. 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 (Cat. No. KHO0101) and AKT (Phospho) [pS473] Human ELISA Kit (Cat. No. KHO0111) 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 2. 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 (Cat. No. KHO0111). The graph shows that only the peptide corresponding to the region surrounding serine 473 blocks the ELISA signal.

Figure 3. 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 (Cat. No. KHO0111) 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].

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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 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 4), SMAD1 (Figure 5) and evaluated for specificity by immunoblot analysis. Few examples of published data using InstantOne Phospho-ELISA kits have been highlighted in Table 1.

Benefits of InstantOne Phospho-ELISA kits:

• Flexible—enables the detection of total and/or phospho protein levels of a single protein up or down a pathway on a single plate
• Specificity—two antibodies directed against the analyte provide better specificity than western blotting
• Sensitivity—more sensitive than western blotting with only typically 2,000-3,000 cells needed
• High throughput—1 hour and 1 wash protocol

InstantOne Phospho-ELISA kit contents:

Explore InstantOne Phospho-ELISA kits

Figure 4. 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 immunoblotting (right) and ELISA (left) using p38 MAPK (Phospho) [pT180/pY182] Multispecies InstantOne ELISA Kit (Cat. No. 85-86022-11). The intensity of the band at 38 kDa as detected by immunoblot correlates with InstantOne ELISA absorption values.

Figure 5. 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 (Cat. No. 85-86183-11).

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12. Miyagishima K.J., Sharma R., Nimmagadda M., et al. AMPK modulation ameliorates dominant disease phenotypes of CTRP5 variant in retinal degeneration. Commun Biol, 2021. 4(1): 1360. 
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