ERK Phosphorylation Assay Kit

Catalog No. ABIN1019677

Product Details

Target: ERK2 (MAPK1) (Mitogen-Activated Protein Kinase 1 (MAPK1))

Binding Specificity: phosphorylated

Reactivity: Human, Rat, Mouse

Application: Biochemical Assay (BCA)

Characteristics: Safe. Non-radioactive assay. Simple and convenient. Total and pERK can be measured in the same sample

Components: 10x Wash Buffer: 25 mL. Blocking Buffer: 25 mL. ALP Substrate: 6 mL. HRP Substrate: 6 mL. ERK-Ab1: 10 µL. pERK-Ab1 10 µL. HRP-Ab2: 10 µL. ALP-Ab2 10 µL

Material not included: 37% formaldehyde, 3% H2O2, black cell culture 96-well plate, plate sealers, deionized or distilled water, pipetting devices, cell culture incubators, centrifuge tubes, fluorescence plate reader capable of reading at gamma ex/em = 535/590nm and at gamma ex/em =360/450nm.

Application Details

Application Notes: Determination of ERK phosphorylation status in whole cells. Evalutation of effects of ligands or drugs on ERK phosphorylation.

Protocol: Important:
1. To avoid cross-contamination, change pipette tips between additions of each reagent or sample. Use separate reservoirs for each reagent. Prior to Assay, dilute 10x Wash Buffer in dH2O to prepare 250 mL 1x Wash Buffer.
2. It is recommended that assays be run in duplicate. Plan to use four assay wells for each sample: two Sample Background wells in which Blocking Buffer is added for determining Ab2 background fluorescence, and two Sample wells in which Ab1 Mixture is added (see Step B2).

A. Culture and Treat Cells
1. Seed 100 µL of 2-4 - 10^4 adherent cells (or 4-10x 10 4 suspension cells) into each well of a black clear-bottom 96-well plate. Incubate overnight at 37° C in a cell culture incubator. Note: The cell number to be used depends on the cell line and ERK1/2 phosphorylation status.
2. Treat the cells as desired (e.g. with ligands or drugs).
3. Prepare formaldehyde solutions (warning: formaldehyde is toxic. Use chemical hood and wear appropriate gloves and eye protection): For adherent cells, prepare 4% formaldehyde by mixing 1.3 mL of 37% formaldehyde and 10.7 mL of 1x Wash buffer. Simply fix cells in each well by replacing the medium with 100 µL of 4% formaldehyde. For suspension cells, prepare 8% formaldehyde by mixing 2.6 mL of 37% formaldehyde and 9.4 mL of 1x Wash buffer. Centrifuge the plate at 500g for 15 min at 4° C and carefully remove as much media as possible without disturbing the cell pellet (repeat this for suspension cells with each wash step below). Fix the cells in each well by adding 100 µL of 8% formaldehyde to cell pellet. Cover the plate and incubate for 20 min at room temperature. Alternatively, the plate containing the fixed cells can be sealed and stored for up to 2 weeks at 2-8° C.
4. Remove the formaldehyde solution and wash the cells 3 times with 150 µL of 1x Wash Buffer. Each wash step should be performed for 5 min with gentle shaking.
5. Prepare Quench Buffer by mixing 2.2 mL of 3% H2O2 and 8.8 mL of 1x Wash Buffer. Remove the Wash Buffer and add 100 µL of Quench Buffer to each assay well. Cover plate and incubate for 20 min at room temperature.
6. Remove the Quench Buffer and wash the cells 3 times with 150 µL of 1x Wash Buffer. Each wash step should be performed for 5 min with gentle shaking.
7. Remove the Wash Buffer, and add 100 µL of Blocking Buffer. Cover plate and incubate for 1 hr at room temperature.

B. Add Primary Antibodies (Ab1)
1. Add 100 µL of PBS to the ERK-Ab1 and pERK-Ab1 tubes and mix well. Prepare enough primary antibody Ab1 Mixture for each well by mixing 1 µL diluted ERK-Ab1, 1 µL diluted pERK-Ab1 and 55 µL Blocking Buffer. Unused Ab1 antibodies can be stored at -20° C for up to 45 days.
2. Remove the Blocking Buffer from all assay wells. Add 50 µL of the Blocking Buffer to the Sample Background wells and 50 µL of Ab1 Mixture to the Sample wells. Cover plate and incubate for 3 hrs at room temperature or overnight at 2-8° C with gentle shaking.
3. Remove the Ab1 Mixture and wash the cells 3 times with 150 µL of 1x Wash Buffer. Each wash step should be performed for 5 min with gentle shaking.

C. Add Secondary Antibodies (Ab2)
1. Immediately before use, add 100 µL of PBS to the HRP-Ab2 and ALP- Ab2 tubes and mix well. Prepare enough secondary antibody Ab2 Mixture, for each well, by mixing 1 µL diluted HRP-Ab2, 1 µL diluted ALP-Ab2 and 55 µL Blocking Buffer. Unused Ab2 antibodies can be stored at -20° C for up to 45 days.
2. Remove Wash Buffer and add 50 µL of the Ab2 Mixture to all assay wells. Cover plate and incubate for 2 hrs at room temperature with gentle shaking.

D. Detection
1. Remove the Ab2 Mixture from each well and wash the cells 5 times with 150 µL of 1x Wash Buffer. Each wash step should be performed for 5 min with gentle shaking.
2. Immediately before use, add 6 µL 3% H2O2 to the provided 6 mL HRP Substrate (for partial plate assay, adjust the volumes accordingly). Remove the Wash Buffer from the plate and add 50 µL of reconstituted HRP Substrate to each well. Incubate for 20 min at room temperature in the dark.
3. Add 50 µL of ALP Substrate to each well and incubate for an additional 20 min at room temperature in the dark.
4. Read the plate at ex/em = 535/590nm for phosphorylated ERK (pERK) and at ex/em =360/450nm for total ERK (ERK).

Calculation of Results:

Calculate the mean fluorescence intensities for the Sample Background wells and Sample wells. Subtract the mean fluorescence of the Sample Background wells from the fluorescence value of the Sample well to yield F values for the phosphorylated ERK (FpERK) at 535/590nm and the total ERK (FERK) at 360/450nm.

Restrictions: For Research Use only

Handling

Storage: -20 °C

Target Details

Target: ERK2 (MAPK1) (Mitogen-Activated Protein Kinase 1 (MAPK1))

Alternative Name: ERK (MAPK1 Products)

Synonyms: ERK-1 Kit, ERK1 Kit, ERT2 Kit, HS44KDAP Kit, HUMKER1A Kit, P44ERK1 Kit, P44MAPK Kit, PRKM3 Kit, p44-ERK1 Kit, p44-MAPK Kit, Erk-1 Kit, Erk1 Kit, Ert2 Kit, Esrk1 Kit, Mnk1 Kit, Mtap2k Kit, Prkm3 Kit, p44 Kit, p44erk1 Kit, p44mapk Kit, ERK Kit, ERK2 Kit, ERT1 Kit, MAPK2 Kit, P42MAPK Kit, PRKM1 Kit, PRKM2 Kit, p38 Kit, p40 Kit, p41 Kit, p41mapk Kit, 9030612K14Rik Kit, AA407128 Kit, AU018647 Kit, C78273 Kit, Erk2 Kit, Prkm1 Kit, p42mapk Kit, zERK2 Kit, MAPK1 Kit, Xp42 Kit, erk Kit, erk2 Kit, ert1 Kit, mapk Kit, mapk1b Kit, mapk2 Kit, mpk1 Kit, prkm1 Kit, prkm2 Kit, mapk1 Kit, mapk1a Kit, xp42 Kit, ATMPK1 Kit, F14N23.9 Kit, F14N23_9 Kit, MITOGEN-ACTIVATED PROTEIN KINASE Kit, mitogen-activated protein kinase 1 Kit, MNK1 Kit, ATMPK2 Kit, MITOGEN-ACTIVATED PROTEIN KINASE HOMOLOG 2 Kit, T30E16.13 Kit, T30E16_13 Kit, mitogen-activated protein kinase homolog 2 Kit, mitogen-activated protein kinase 3 Kit, mitogen-activated protein kinase 1 Kit, mitogen activated protein kinase 1 Kit, mitogen-activated protein kinase 1 L homeolog Kit, mitogen-activated protein kinase 1 S homeolog Kit, mitogen activated protein kinase 3 Kit, mitogen-activated protein kinase homolog 2 Kit, MAPK3 Kit, Mapk3 Kit, MAPK1 Kit, Mapk1 Kit, mapk1 Kit, mapk1.L Kit, mapk1.S Kit, PfMAP1 Kit, MPK1 Kit, PVX_084965 Kit, LOC9328617 Kit, Tsp_01601 Kit, MPK2 Kit

Background: Quantitative fluorescent immunoenzymatic assay for ERK1/2 phosphorylation status in cultured cells. This simple assay eliminates the need for cell lysate preparation and is used to study kinase signaling and the effects of kinase inhibitors on cells.

The mitogen-activated protein kinase (MAPK/ERK) pathway plays a key role in cell proliferation, differentiation and migration. Stimulation by mitogens eventually leads to phosphorylation of ERK1 (T202/Y204) and ERK2 (T185/Y187). The MAPK/ERK cascade presents many interesting drug targets for the development of cancer therapies. This cell-based ELISA measures dually phosphorylated ERK1/2 in whole cells. This simple and efficient assay eliminates the need for cell lysate preparation and can be used to study kinase signaling and the effects of kinase inhibitors on cells. In this assay, cells are grown in 96- well plates and treated with ligands or drugs. Cells are then fixed and permeabilized in the wells. ERK1/2 phosphorylation (pERK) is measured using a double immunoenzymatic labeling procedure. Principle of Cell-Based ERK ELISA A. Culture & treat cells B. Add Ab1 C. Add Ab2 D. Add fluorogenic substrates. Measure fluorescence intensity.mouse anti-phosphoERK anti-rabbit IgG-ALPanti-rabbit IgG-ALP anti-mouse IgG-HRPanti-mouse IgG-HRP rabbit anti-total ERKPrimary Ab1: Secondary Ab2: ALP (360/450nm) pERK ERKpERK ERK pERK ERKpERK ERK Y Y Y pERK ERKpERK ERK YY Y pERK ERKpERK ERK Y Y Y HRP (535/590nm