Description
Key facts
Detection method:Colorimetric,
Sample types:Tissue Homogenate, Cell Lysate,
Reacts with:Mouse, Human,
Assay type:Semi-quantitative,
Assay time:1h 30m,
Assay Platform:Microplate
Product details:
ERK1 (phospho T202 + Y204) + ERK2 (phospho T185 + Y187) + Total ELISA Kit ab176660 is a semi-quantitative sandwich ELISA to measure ERK1 (phospho T202 + Y204) + ERK2 (phospho T185 + Y187) + Total in cell lysates.
How the assay works:
The SimpleStep ELISA™ employs an affinity tag labeled capture antibody and a reporter conjugated detector antibody which immunocapture the sample analyte in solution. This entire complex (capture antibody/analyte/detector antibody) is in turn immobilized via immunoaffinity of an anti-tag antibody coating the well. To perform the assay, samples or standards are added to the wells, followed by the antibody mix. After incubation, the wells are washed to remove unbound material. TMB substrate is added and during incubation is catalyzed by HRP, generating blue coloration. This reaction is then stopped by addition of Stop Solution completing any color change from blue to yellow. Signal is generated proportionally to the amount of bound analyte and the intensity is measured at 450 nm. Optionally, instead of the endpoint reading, development of TMB can be recorded kinetically at 600 nm.
Assay Specificity
The ERK1/2 (pT202/Y204) assay detects endogenous levels of ERK1/2 (GenBank Accessions NP 002737.2 [ERK1], NP 620407 [ERK2]) in cellular lysates, only when phosphorylated at Thr202/Tyr204.
The ERK1/2 Total assay detects endogenous levels of ERK1/2 (GenBank Accessions NP 002737.2 [ERK1], NP 620407 [ERK2]) in cellular lysates, irrespective of phosphorylation status.
ERK1 (phospho T202 + Y204) + ERK2 (phospho T185 + Y187) + Total ELISA Kit ab176660 protocol summary
1. Add 50 µL control or sample to appropriate wells
2. Add 50 µL Antibody Cocktail to all wells. Incubate at room temperature for 1 hour
3. Aspirate and wash each well three times with 350 µL 1X Wash Buffer PT
4. Add 100 µL TMB Substrate to each well and incubate for 15 minutes.
5. Add 100 µL Stop Solution and read OD at 450 nm
Estimated sensitivity
: Phospho-ERK (Thr202/Tyr204): 0.1 ng/mL (tested with recombinant protein), Total ERK1/2: 0.6 ng/mL (tested with recombinant protein)
: Phospho-ERK (Thr202/Tyr204): 0.2-20ng/mL, Total ERK1/2: 0.6-60 ng/mL
REACH authorisation
Abcam has not and does not intend to apply for the REACH Authorisation of customers' uses of products that contain European Authorisation list (Annex XIV) substances.
It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.
Properties and Storage Information:
Shipped at conditions-Blue Ice, Appropriate short-term storage conditions-+4°C, Appropriate long-term storage conditions-+4°C, Storage information-+4°C
Supplementary Information:
This supplementary information is collated from multiple sources and compiled automatically.
ERK1 and ERK2 also known as p44 and p42 MAPK respectively are important proteins in the MAP kinase signaling pathway. They are expressed in various tissues with significant presence in the brain lungs and skin. ERK1 has a molecular weight of approximately 44 kDa while ERK2 has a molecular weight of around 42 kDa. Both proteins become activated through phosphorylation which is essential for their function in cellular processes.
Biological function summary
ERK1 and ERK2 serve as key players in cellular growth differentiation and survival. They form part of a complex cascade where they transduce signals from the cell membrane to the nucleus after activation by phosphorylation. This phosphorylation enables them to modify various downstream targets involved in regulating gene expression and cellular response to external stimuli.
Pathways
ERK1 and ERK2 are critical components of the MAPK/ERK pathway and the Ras-Raf-MEK-ERK signaling cascade. These pathways regulate a multitude of cellular activities including proliferation and differentiation. In the MAPK/ERK pathway proteins like Ras and Raf serve as upstream activators of ERK1 and ERK2. Both ERK1 and ERK2 also interact with other signaling proteins such as MEK1/2 which directly phosphorylates and activates them.
Dysregulation of ERK1 and ERK2 is associated with various pathologies including cancer and neurodegenerative diseases. Abnormal activation of these proteins often leads to uncontrolled cell proliferation contributing to oncogenesis. For instance mutations in proteins like Ras which regulate ERK1 and ERK2 can result in continuous activation and lead to tumor formation. Furthermore altered ERK1 and ERK2 signaling is linked to neurodegeneration impacting neuronal survival and function.
