A convenient cell-based ELISA to measure both total and phosphorylated proteins in a single well

Dual-Detect CELISA is a simple and efficient cell-based ELISA assay to measure both total and phosphorylated targets in whole cells in a 96 well microplate format using dual fluorescent detection. Cells are cultured directly in the microplate, stimulated as desired, fixed, and simultaneously incubated with two primary antibodies: a phospho-specific antibody and a total antibody that recognizes the target-protein, regardless of phosphorylation status. Both the total target protein and the phosphorylated protein is measured in a single well using a double immunoenzymatic labeling procedure, either horseradish-peroxidase (HRP) or alkaline phosphatase (AP), and two spectrally distinct fluorogenic substrates for HRP and AP. The fluorescence of the phosphorylated protein is normalized to the total protein in each well thus reducing well-to-well variations.

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Figure 1: Dual Detect CELISA Fluorescent Cell-based Assay

The Dual Detect CELISA Assay kits contain the components required to run the CELISA using fluorgenic substrates to measure both total phosphorylated target protein in the context of whole cells. Just add cells, treat, and run the assay.

Figure 2: Phospho-AKT (Ser473) Dual Detect CELISA

NIH3T3 cells were seeded at 1x10⁴ cells per well, cultured for 24 hr, serum starved 16 hr, and stimulated with 50ng/mL of PDGF for 0, 1, 5, 15, or 30 minutes. Total and phospho-AKT levels were determined using AKT (Ser473) Dual Detect CELISA Kit. Phosphorylated AKT levels have been normalized to the total AKT.

Insert shows the corresponding Western Blot Data. Lysates were prepared from the NIH3T3 stimulated cells, resolved by electrophoresis and western blotted. AKT was detected using antibodies to either total AKT or phospho-AKT (Ser473).

Figure 3: Phospho-EGFR (Tyr1173) Dual Detect CELISA

A431 cells were seeded at 1x10⁴ cells per well, cultured for 24 hr, serum starved 16 hr, and stimulated with 100ng/mL of EGF for 0, 1, 5, 15, or 30 minutes. Total and phospho-EGFR levels were determined using EGFR (Tyr1173) Dual Detect CELISA Kit. Phosphorylated EGFR levels have been normalized to the total EGFR.

Insert shows the corresponding Western Blot Data. Lysates were prepared from the A431 stimulated cells, resolved by electrophoresis and western blotted. EGFR was detected using antibodies to either total EGFR or phospho-EGFR (Tyr1173).

Figure 4: Phospho-p38 (Thr180/Tyr182) Dual Detect CELISA

HeLa cells were seeded at 1x10⁴ cells per well, cultured for 24 hr, serum starved 16 hr, and stimulated with 25 µg/mL of anisomycin for 0, 5, 15, 30 or 60 minutes. Total and phospho-p38 levels were determined using p38 (Thr180/Tyr182) Dual Detect CELISA Kit. Phosphorylated p38 levels have been normalized to the total p38.

Insert shows the corresponding Western Blot Data. Lysates were prepared from the HeLa stimulated cells, resolved by electrophoresis and western blotted. p38 was detected using antibodies to either total p38 or phospho-p38 (Thr180/Tyr182).

Figure 5: Phospho Erk1/2 (Thr185/Tyr187) Dual-Detect CELISA

NIH3T3 cells were seeded at 1x10⁴ cells per well, cultured for 24 hr, serum starved 16 hr, and stimulated with 50ng/mL of PDGF for 0, 1, 5, 15, or 30 minutes. Total and phospho Erk1/2 levels were determined using Erk (Thr185/Tyr187) Dual-Detect CELISA Kit. Phosphorylated Erk1/2 levels have been normalized to the total Erk.  

Insert shows the corresponding Western Blot Data. Lysates were prepared from the NIH3T3 stimulated cells, resolved by electrophoresis and western blotted. Erk1/2 was detected using antibodies to either total Erk1/2 or phospho Erk (Thr185/Tyr187).

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