Phosphorylation ELISA Kits

Semi-Quantitative, Sandwich ELISAs for Detection of Phosphorylated Proteins in Cell Lysates

The RayBio® Phosphorylation ELISA kits are very rapid, convenient and sensitive assays that monitor the activation or function of important biological pathways in cell lysate samples. By determining phosphorylated protein in experimental model systems, researchers can verify pathway activation and simultaneously measure numerous different cell lysates without spending excess time and effort in performing Western Blot analyses.
Blue Ribbon Seal ELISA

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  • Simultaneously measure Phosphorylated protein and pan protein in one experiment (for normalization purpose)
  • Screen numerous different cell lysates without performing a Western Blot analysis
  • Minimal hands-on time, convenient, and non-radioactive material
  • High sensitivity
  • High quality
  • Cost-effective
  • Complete kit with pre-coated 96 well plate

Contents of Kit

  • 96 wells (12 strips x 8 wells) Coated With Antibody
  • Wash Buffer Concentrate (20x)
  • Positive Control
  • Assay Diluent
  • Detection Antibody
  • Secondary Antibody or HRP Streptavidin
  • TMB One-Step Substrate Reagent
  • Stop Solution
  • Cell Lysis Buffer

Pick-Your-Pathway Program

Interested in phosphorlyation and cell signaling signaling pathways? RayBiotech now offers a Pick-Your-Pathway Program which allows you to quickly select your own panel of signaling molecules to create a custom multi-kit phosphorylation ELISA. Simply choose any 2 or more of our in-stock, Phosphorylation ELISA kits, and we will combine them into a multi-box kit for you. This program has a great discount built in, allowing you to perform pathway analysis while saving money.

To learn more about this program, click the button below:


How It Works

Phospho-ELISA - How it works

This Sandwich ELISA kit is an in vitro enzyme-linked immunosorbent assay for the measurement of phosphorylated protein. An specific capture antibody (anti-pan-antibody or anti-phosphorylated antibody) has been coated onto a 96-well plate. Samples are pipetted into the wells and target protein present in a sample is bound to the wells by the immobilized antibody. The wells are washed and anti-phosphorylated antibody or anti-pan-antibody is used to detect target protein. After washing away unbound antibody, secondary antibody is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of target bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.

GSK3b Positive Control

Representative Results

1. Recombinant Human EGF Stimulation of A431 Cell Lines:

A431 cells were treated or untreated with 100 ng/ml recombinant human EGF for 10 min. Cell lysates were analyzed using this phosphoELISA and Western Blot.

Phospho ELISA

2. Sensitivity:

The A431 cells were treated with 100 ng/mL recombinant human EGF for 20 minutes to induce phosphorylation of EGF R. Serial dilutions of lysates were analyzed in this ELISA and by Western blot. Immunoblots were incubated with anti-phospho-EGFR (Tyr 1086).

Phospho ELISA Sensitivity

Reference List

  1. Profiling of cytokine expression by biotin-labeled-based proteinarrays. Ying Lin, Ruochun Huang, Lipai Chen et al. Proteomics.2003, 3: 1750-1757.
  2. Proteomic profiling of the cancer microenvironment by antibody arrays. Vladimir Knezevic, Chidchanok Leethanakul, Verena E. Bichsel et al. Proteomics 2001, 1, 1271“1278
  3. Antibody microarray profiling of human prostate cancer sera: Antibody screening and identification of potential biomarkers Jeremy C. Miller, Heping Zhou, Joshua Kwekel, Robert Cavalloet al. The, Brian B. Haab. Proteomics. 2003, 3: 56-63.
  4. Smad4 signalling in T cells is required for suppression of gastrointestinal cancer. Byung-Gyu Kim, Cuiling Li, WenhuiQiao, Mizuko Mamura, Barbara Kasperczak, Miriam Anver, Lawrence Wolfraim, Suntaek Hong, Elizabeth Mushinski, Michael Potter, Seong-Jin Kim, Xin-Yuan Fu, Chuxia Deng and John J. Letterio. Nature. 2006; Vol 441: 1015
  5. Connexin suppresses human glioblastoma cell growth by down-regulation of monocyte chemotactic protein 1, as discoveredusing protein array technology. Ruochun Huang, Ying Lin, Cheng C. Wang, Jacob Gano, Biaoyang Lin, Qian Shi, AltonBoynton, Jocelyn Burke, and Ruo-Pan Huang. Cancer Res. 2002;62:2806-2812.
  6. LPS induces the interaction of a transcription factor, LPS-induced TNF-a factor, and STAT6(B) with effects on multiple cytokines.Xiaoren Tang, Deborah Levy Marciano, Susan E. Leeman and Salomon Amar. PNAS. April 5, 2005 vol. 102 no. 14, 5132-5137
  7. HIV-1-mediated apoptosis of neuronal cells: Proximal molecular mechanisms of HIV-1-induced encephalopathy. Yan Xu, JosephKulkoshy, Roger j. Pomerantz. PNAS. 2004 May 4, 2004 Vol. 101 No. 18.
  8. A novel method for high-throughput protein profiling fromconditioned media and patient's sera. Ruo-Pan Huang, RuochunHuang, Yan Fan, and Ying Lin. Ana. Biochem. 2001;294(1):55-62.