Measure signaling of intracellular proteins

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

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 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:

  • Pre-coated 96 well plate
  • Detection Antibody
  • Standard
  • Standard Diluent Buffer
  • Anti-rabbit IgG HRP
  • HRP Diluent
  • Wash Buffer
  • Chromogen (TMB)
  • Stop Solution
  • Adhesive Plate Covers
Representative data showing total vs. phosphorylated AKT in Jurkat cells

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.

Line plot of AKT [pS473] specificity using peptide competition assay

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.

Representative data showing sensitivity of AKT [pS473] ELISA

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].

How to use Invitrogen precoated Phospho-ELISA kits

View the instructional video that demonstrates how to use the precoated sandwich ELISA kit for phospho-p38. Please carefully read and review your ELISA kit’s specific instruction sheet, located inside the package, for detailed information before starting the experiment.

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 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:

  • InstantOne Assay Plate
  • Capture Antibody
  • Detection Antibody
  • Cell Lysis Buffer
  • Enhancer Solution
  • Wash Buffer
  • Detection Reagent (TMB Substrate)
  • Stop Solution
  • Positive Control Cell Lysate (lyophilized)
  • Plate seal
Detection of p38 MAPK (phospho) in anisomycin-treated HEK293 cells

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.

Detection of total and phosphorylated SMAD1 in stimulated C2C12 cells.

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).

Table 1. List of publications highlighting Phospho-ELISA kits used in research studies.

ReferenceSummaryTarget protein/sPhospho-ELISA Kits
Vallés-Martí et al. 2023 In-depth phosphoproteomics study for identification of hyperactive kinases in pancreatic ductal adenocarcinoma (PDAC).EGFREGFR (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.TauTau (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.CREBCREB (Total) Human ELISA Kit
CREB (Phospho)

CREB (Phospho) [pS133] Human ELISA Kit

Tamura et al. 2023 Somatostatin receptor (SSTR5) antagonists may represent a novel and attractive therapeutic agent for type 2 diabetes.AKTAKT (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-kBNF-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)

IRS1 (Phospho) [pS312] Human ELISA Kit

Additional ELISA products

Cell Signaling Pathways brochure

Explore our antibodies for cell signaling pathways for different areas of biology, including angiogenesis, apoptosis, bone biology, metabolism, transcription factors, and more.

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  1. Vallés-Martí A., Mantini G., Manoukian P., et al. Phosphoproteomics guides effective low-dose drug combinations against pancreatic ductal adenocarcinoma. Cell Rep, 2023. 42(6): 112581. 
  2. Walker C.K., Greathouse K.M., Tuscher J.J., et al. Cross-Platform Synaptic Network Analysis of Human Entorhinal Cortex Identifies TWF2 as a Modulator of Dendritic Spine Length. J Neurosci, 2023. 43(20): 3764-3785 
  3. Scuruchi M., Aliquò F., Avenoso A., et al. Endocan Knockdown Down-Regulates the Expression of Angiogenesis-Associated Genes in Il-1ß Activated Chondrocytes. Biomolecules, 2023. 13(5): 851 
  4. Inostroza-Nieves Y., Rivera A., Romero J.R. Blockade of endothelin-1 receptor B regulates molecules of the major histocompatibility complex in sickle cell disease. Front Immunol, 2023. 14: 1124269. 
  5. Tamura Y.O., Sugama J., Abe S.I., et al. Selective somatostatin receptor 5 inhibition improves hepatic insulin sensitivity. Pharmacol Res Perspect, 2023. 11(1): e01043. 
  6. An J. and Cho J. Effect of long-chain inorganic polyphosphate treated with wheat phytase on interleukin 8 signaling in HT-29 cells. Anim Biosci, 2022. 35(6): 892-901. 
  7. Cornwell A., Fedotova S., Cowan S., et al. Cystathionine γ-lyase and hydrogen sulfide modulates glucose transporter Glut1 expression via NF-κB and PI3k/Akt in macrophages during inflammation. PLoS One, 2022. 17(12): e0278910. 
  8. Wang X., Chen G., Nie L., et al. IL-2K35C-moFA, a Long-Acting Engineered Cytokine with Decreased Interleukin 2 Receptor α Binding, Improved the Cellular Selectivity Profile and Antitumor Efficacy in a Mouse Tumor Model. Cancers (Basel), 2022. 14(19): 4742 
  9. Marfella R., D'Onofrio N., Trotta M.C., et al. Sodium/glucose cotransporter 2 (SGLT2) inhibitors improve cardiac function by reducing JunD expression in human diabetic hearts. Metabolism, 2022. 127: 154936. 
  10. Oliveira-Santos A., Dagda M., Burkin D.J. Sunitinib inhibits STAT3 phosphorylation in cardiac muscle and prevents cardiomyopathy in the mdx mouse model of Duchenne muscular dystrophy. Hum Mol Genet, 2022. 31(14): 2358-2369 
  11. Okabe S., Tanaka Y., Gotoh A. Therapeutic targeting of PFKFB3 and PFKFB4 in multiple myeloma cells under hypoxic conditions. Biomark Res, 2022. 10(1): 31. 
  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. 
  13. Nasca C., Dobbin J., Bigio B., et al. Insulin receptor substrate in brain-enriched exosomes in subjects with major depression: on the path of creation of biosignatures of central insulin resistance. Mol Psychiatry, 2021. 26(9): 5140-5149. 

For Research Use Only. Not for use in diagnostic procedures.

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