Signaling Pathway Assays

ERK Phosphorylation Assay Kit (BA0098)

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SKU:
BA0098
Product Type:
Assay
Research Area:
Cell Biology
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ERK Phosphorylation Assay Kit (BA0098) (BA0098)

The ERK Phosphorylation Assay Kit (SKU: BA0098) is a fluorimetric, cell-based ELISA that measures dually phosphorylated ERK1/2 in whole cells and normalises the signal to total protein content. The MAPK/ERK pathway plays a key role in cell proliferation, differentiation and migration, with stimulation by mitogens leading to phosphorylation of ERK1 (T202/Y204) and ERK2 (T185/Y187), and it presents many attractive drug targets for cancer therapy. This simple and efficient assay eliminates the need for cell lysate preparation and can be used to study kinase signalling and the effects of kinase inhibitors on cells. Cells are grown in 96-well plates, treated with ligands or drugs, then fixed and permeabilised in the wells, after which ERK1/2 phosphorylation is measured by fluorescent ELISA followed by total protein measurement in each well.

Product Name:ERK Phosphorylation Assay Kit (BA0098)
SKU:BA0098
Detection Method:Fluorometric cell-based ELISA
Detection Range:Normalised phosphorylated ERK relative to a control reference value (e.g. untreated or time-zero wells)
Sample Type:Cultured adherent or suspension cells in 96-well plates
Species Reactivity:Human, mouse and rat
Assay Time:Total assay time about 6.5 hours (hands-on time 2.5 hours)
Kit Size:100 Assays
Equipment Required:Microplate reader
Storage:-20C
Shelf Life:6 months after receipt
Shipping:Gel Pack

Fluorimetric cell-based ELISA for ERK1/2 phosphorylation status. Cells are cultured and treated in 96-well plates, fixed and permeabilised, then dually phosphorylated ERK1/2 is detected by fluorescent ELISA (530/585 nm) and normalised to total cellular protein (360/450 nm) in the same well.

  • New and improved; total assay time reduced from 21 hours to 6.5 hours (hands-on time 2.5 hours)
  • Simple and convenient; cells are cultured directly in 96-well plates with no cell lysis required
  • Accurate and high-throughput; phosphorylation is normalised to total cellular protein in the same well, minimising well-to-well variation
  • Readily automated for thousands of samples per day
  • Quantitative fluorescent immunoenzymatic assay of ERK1/2 phosphorylation status in cultured cells
  • Evaluation of the effects of ligands or drugs on ERK phosphorylation

Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

StepProcedure
1Preparation. Change pipette tips between reagents and use a multi-channel pipette. Prepare 1x Wash Buffer by diluting Stock Wash Buffer 40-fold with distilled water (e.g. 10 mL Stock Wash Buffer plus 390 mL distilled water) and reserve 6 mL for the detection step. Assay samples in triplicate or higher, and include a Protein Blank (no cells) and a Sample Blank (cells with only Ab2) in triplicate for each plate and sample.
2Culture and treat cells. Seed 100 uL of 2-4 x 10^4 adherent cells (or 4-10 x 10^4 suspension cells) into each well of a black 96-well culture plate, adding 100 uL culture media without cells into three wells for the Protein Blank, and incubate overnight at 37C. Treat the cells as desired with ligands or drugs.
3Fix and permeabilise. Prepare 4% formaldehyde for adherent cells (or 8% for suspension cells) and fix the cells in each well; incubate for 20 minutes at room temperature. Remove the formaldehyde and wash three times with 150 uL 1x Wash Buffer. Prepare Quench Buffer by mixing 2.2 mL 3% H2O2 and 8.8 mL 1x Wash Buffer, add 100 uL per well, incubate 20 minutes at room temperature and wash three times. Add 100 uL Blocking Buffer and incubate 1 hour at room temperature.
4Add primary antibody (Ab1). Prepare 55 uL Ab1 mixture per well by diluting Ab1 1:625 in Blocking Buffer. Remove the wash buffer, add 50 uL Blocking Buffer to the Sample Blank wells and 50 uL Ab1 mixture to the Sample wells, and incubate 90 minutes at room temperature (or overnight at 2-8C) with gentle shaking. Wash three times with 150 uL 1x Wash Buffer.
5Add secondary antibody (Ab2). Prepare 55 uL Ab2 mixture per well by diluting Ab2 1:625 in Blocking Buffer. Remove the wash buffer, add 50 uL Ab2 mixture to all assay wells and incubate 90 minutes at room temperature with gentle shaking.
6Detection. Remove the Ab2 mixture and wash five times with 150 uL 1x Wash Buffer. Prepare HRP Substrate by mixing 60 uL Dye Reagent with 6 mL 1x Wash Buffer and 6 uL 3% H2O2, add 50 uL per well and incubate 30 minutes at room temperature in the dark. Add 50 uL Protein Stain per well and incubate a further 5 minutes in the dark.
7Read the plate at excitation/emission 530/585 nm for phosphorylated ERK (pERK) and at 360/450 nm for total protein.

Calculate the mean pERK fluorescence at 530/585 nm for the Sample Blank wells (F-BLK pERK) and Sample wells (F-SAMPLE pERK), and the mean protein fluorescence at 360/450 nm for the Protein Blank well (F-BLK Prot) and Sample wells (F-SAMPLE Prot). Subtract the Sample Blank pERK fluorescence from the Sample pERK fluorescence to give dF-pERK, and subtract the Protein Blank protein fluorescence from the Sample protein fluorescence to give dF-Prot. Normalised phosphorylated ERK = (dF-pERK / dF-Prot) divided by the control reference value (dF-pERK / dF-Prot)o, where the reference is a control such as time zero in kinetic studies or untreated wells in drug potency studies.

ComponentQuantityStorage
Stock Wash Buffer25 mL-20C
Blocking Buffer25 mL-20C
Protein Stain6 mL-20C
Dye Reagent120 uL-20C
Ab110 uL-20C
Ab2 (gM-HRP)10 uL-20C
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