Human/Mouse/Rat Phospho-ERK1 (T202/Y204) DuoSet IC ELISA

Name: Human/Mouse/Rat Phospho-ERK1 (T202/Y204) DuoSet IC ELISA
Brand: R&D Systems
SKU: DYC1825-2
Price: 495 USD
Availability: InStock

R&D Systems | Catalog # DYC1825-2

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Product Summary
Product Specifications
Scientific Data
Kit Contents
Other Reagents Required
Preparation & Storage
Background
Product Documents
Specific Notices
Research Areas

Key Product Details

Assay Type

Solid Phase Sandwich ELISA

Assay Range

125-8000 pg/mL

Sample Type

Cell Lysates

Reactivity

Human, Mouse, Rat

Human/Mouse/Rat Phospho-ERK1 (T202/Y204) DuoSet IC ELISA Features

Optimized capture and detection antibody pairings with recommended concentrations save lengthy development time
Development protocols are provided to guide further assay optimization
Assay can be customized to your specific needs
Available in 2, 5, and 15- (96-well) plate pack sizes
Economical alternative to Western blot

View all ERK1 ELISA Kits »

Product Summary for Human/Mouse/Rat Phospho-ERK1 (T202/Y204) DuoSet IC ELISA

This DuoSet IC ELISA contains the basic components required for the development of sandwich ELISAs to measure phosphorylated human, mouse, and rat ERK1 (T202 / Y204) in cell lysates. An immobilized capture antibody specific for ERK1 binds both phosphorylated and unphosphorylated ERK1. After washing away unbound material, a biotinylated detection antibody is used to detect only phosphorylated receptor, utilizing a standard HRP format.

Product Specifications

Assay Format

96-well strip plate (sold separately)

Sample Volume Required

Cell lysates (100 µL)

Detection Method

Colorimetric ELISA - 450nm (TMB)

Conjugate

Biotin

Specificity

Please see the product datasheet

Label

HRP

Scientific Data Images for Human/Mouse/Rat Phospho-ERK1 (T202/Y204) DuoSet IC ELISA

Standard Curve

Specificity of Human/Mouse/Rat Phospho-ERK1 (T2002/Y204) DuoSet IC ELISA is Shown by Western Blot.

Lysates prepared from HeLa human cervical epithelial carcinoma cells treated with 200 nM phorbol 12-myristate 13-acetate (PMA) were incubated in wells coated with Phospho-ERK1 Capture Antibody. Unbound material was removed by washing and bound material was solubilized in SDS gel sample buffer. The same lysate and captured proteins were electrophoresed, transferred to a PVDF membrane and immunoblotted with Phospho-ERK1 Detection Antibody. Only a single band corresponding to phosphorylated ERK1 was detected. While present in lysate at greater amounts than ERK1, phosphorylated ERK2 was not present in the captured material.

Quantification of Phosphorylated ERK1 in PMA-treated Human HeLa Cells.

HeLa human cervical epithelial carcinoma cells were treated with 200 nM PMA for the indicated times. Following cell lysis, ERK1 phosphorylated at T202/Y204 was quantified with this DuoSet IC ELISA. The same lysates were also immunoblotted (inset) with either Anti-phospho-ERK1/ERK2 (p-ERK1/ERK2) (Catalog # AF1575) or anti-ERK1 polyclonal antibodies. The DuoSet IC ELISA results correlate well with the relative amounts of phosphorylated ERK1 detected by Western blot (upper molecular weight band of upper blot corresponds to phosphorylated ERK1). The blot with anti-ERK1 antibody indicates that total levels of ERK1 remained constant during incubations with PMA.The quantification of phosphorylated ERK1 with this DuoSet IC ELISA was also determined in cells pretreated with the MEK1/2 inhibitors U0126 and PD98059, which indirectly inhibit phosphorylation of ERK1 at T202/Y204.

Quantification of Phosphorylated ERK1 in U0126- and PD98059-treated Human HeLa Cells.

HeLa human cervical epithelial carcinoma cells were untreated or treated with 200 nM PMA for 20 minutes, either with or without 50 mM U0126 or 50 mM PD98059. Cells were lysed and ERK1 phosphorylated at T202/Y204 was quantified with this DuoSet IC ELISA. The same lysates were also immunoblotted (inset) with either anti-phospho- ERK1/ERK2 (p-ERK1/ERK2) or anti-ERK1 polyclonal antibodies. The DuoSet IC ELISA results correlate well with the total amounts of phosphorylated ERK1 detected by Western blot. The blot with anti-ERK1 antibody indicates that total levels of ERK1 remained constant during the various treatments.This DuoSet IC ELISA also quantifies phosphorylated ERK1 levels in mouse and rat cell lysates.

Quantification of Phosphorylated ERL1 in Growth Factor-treated Mouse and Rat Cells.

Lysates prepared from NIH/3T3 mouse embryonic fibroblast cells either uninduced or induced with 100 ng/mL PDGF (Catalog # 120-HD) (left panel), and PC-12 rat adrenal pheochromocytoma cells either uninduced or induced with 100 ng/mL beta-NGF (Catalog # 556-NG) (right panel) were quantified with this DuoSet IC ELISA. The same lysates were also immunoblotted (inset) with either anti-phospho-ERK1/ERK2 (p-ERK1/ERK2) or anti-ERK1 polyclonal antibodies. The DuoSet IC ELISA results correlate well with the total amounts of phosphorylated ERK1 detected by Western blot. The blots with anti-ERK1 antibody indicate that total levels of ERK1 remained constant during the growth factor inductions.

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Kit Contents for Human/Mouse/Rat Phospho-ERK1 (T202/Y204) DuoSet IC ELISA

Capture Antibody
Conjugated Detection Antibody
Calibrated Immunoassay Standard or Control
Streptavidin-HRP

Other Reagents Required

PBS: (Catalog # DY006), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, 1.5 mM KH₂O₄, pH 7.2 - 7.4, 0.2 µm filtered

Wash Buffer: (Catalog # WA126), or equivalent

Lysis Buffer*

IC Diluent*

Blocking Buffer*

Substrate Solution: 1:1 mixture of Color Reagent A (H₂O₂) and Color Reagent B (Tetramethylbenzidine) (Catalog # DY999)

Stop Solution: 2 N H₂SO₄ (Catalog # DY994)

Microplates: From Costar EIA Plate (Costar Catalog # 2592) or R&D Systems (Catalog # DY990), or equivalent

Plate Sealers: ELISA Plate Sealers (Catalog # DY992), or equivalent

*For the Lysis Buffer, IC Diluent, and Blocking BUffer recommended for a specific DuoSet ELISA Development Kit, please see the product

Preparation and Storage

Shipping

The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store the unopened product at 2 - 8 °C. Do not use past expiration date.

Background: ERK1

ERK1 and ERK2 (also known as MAPK3 and MAPK1) are 44 and 42 kDa Ser/Thr kinases, respectively. They are part of the Ras-Raf-ERK signal transduction cascade often found downstream of growth factor receptor activation. ERK1 and ERK2 were initially isolated and cloned as kinases activated in response to insulin and NGF. They are expressed in most, if not all, mammalian tissues. Dual threonine and tyrosine phosphorylation activate both ERKs, at Thr202/Tyr204 for human ERK1 and Thr185/Tyr187 for human ERK2.

ERK5, also known as Big Mitogen-activated Protein Kinase 1 (BMK1) and MAPK7, is activated by several mechanisms, including receptor tyrosine kinases, G protein-coupled receptors, and osmotic stress. Like ERK1 and ERK2, ERK5 contains the conserved Thr-Glu-Tyr activation motif in its activation loop. Unlike these ERKs, however, ERK5 contains a unique C-terminal domain that regulates its activation and nuclear translocation.