Description: This MILAN8R monoclonal antibody recognizes human and mouse extracellular signal-regulated kinases 1 and 2 (also known as ERK1/2, p44/p42, or MAPK3/1) when phosphorylated on T202/Y204. ERK1/2 belong to a family of conserved serine/threonine protein kinases known as mitogen-activated protein kinases (MAPKs) that are involved in many cellular programs such as proliferation, differentiation, motility, and survival. ERK1/2 signaling is activated in response to numerous extracellular stimuli including mitogens, growth factors, and cytokines. The primary activators of ERK1/2 are MEK1 and MEK2 which act by phosphorylating the activation loop residues T202/Y204 and T185/Y187 in ERK1 and ERK2, respectively. Several downstream targets of ERK1/2 have been identified, including p90RSK and the transcription factor Elk-1. ERK1/2 are negatively regulated by MAPK phosphatases, known as DUSPs or MKPs, as well as by chemical inhibitors of MEK including U0126 and PD98059. Disruption of the ERK pathway is common in many types of cancer.
Specificity of this MILAN8R clone was determined by ELISA, flow cytometry, and western blotting.
Applications Reported: This MILAN8R antibody has been reported for use in intracellular staining followed by flow cytometric analysis.
Applications Tested: This MILAN8R antibody has been pre-titrated and tested by intracellular staining and flow cytometric analysis of stimulated normal human peripheral blood cells or stimulated mouse splenocytes. This can be used at 5 µL (0.06 µg) per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test.
Protocols: We recommend using Protocol C: Two-step protocol: Fixation/Methanol. Protocol A: Two-step protocol: intracellular (cytoplasmic) proteins and Protocol B: One-step protocol: intracellular (nuclear) proteins cannot be used. All Protocols can be found in the "Staining intracellular Antigens for Flow Cytometry Protocol" located in the Best Protocols Section under the Resources tab online.
PerCP-eFluor® 710 emits at 710 nm and is excited with the blue laser (488 nm); it can be used in place of PerCP-Cyanine5.5. We recommend using a 710/50 bandpass filter, however, the 695/40 bandpass filter is an acceptable alternative. Please make sure that your instrument is capable of detecting this fluorochrome.
Fixation: Samples can be stored in IC Fixation Buffer (cat. 00-8222) (100 µL cell sample + 100 µL IC Fixation Buffer) or 1-step Fix/Lyse Solution (cat. 00-5333) for up to 3 days in the dark at 4°C with minimal impact on brightness and FRET efficiency/compensation. Some generalizations regarding fluorophore performance after fixation can be made, but clone specific performance should be determined empirically.
Excitation: 488 nm; Emission: 710 nm; Laser: Blue Laser.
Filtration: 0.2 µm post-manufacturing filtered.
ERK1 and ERK2 are widely expressed and are involved in the regulation of meiosis, mitosis, and postmitotic functions in differentiated cells. Many different stimuli, including growth factors, cytokines, virus infection, ligands for heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors and transforming agents, activate the ERK1 and ERK2 pathways. When growth factors bind to the receptor tyrosine kinase, Ras interacts with Raf, the serine/threonine protein kinase and activates it as well. Once actived, Raf phosphorylates serine residue in 2 further kinases, MEK1/2, which in turn phosphorylates tyrosine/threonine in extracellular-signal regulated kinase (ERK) 1/2. Upon activation, the ERKs either phosphorylate a number of cytoplasmic targets or migrate to the nucleus, where they phosphorylate and activate a number of transcription factors such as c-Fos and Elk-1.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
Protein Aliases: ERK-1; ERK-2; ERK1; ERK2; ERT1; ERT2; Extracellular signal-regulated kinase 1; Extracellular signal-regulated kinase 2; extracellular signal-related kinase 1; Insulin-stimulated MAP2 kinase; MAP kinase 1; MAP kinase 2; MAP kinase 3; MAP kinase isoform p42; MAP kinase isoform p44; MAPK 1; MAPK 2; MAPK 3; MAPK1; MAPK3; Microtubule-associated protein 2 kinase; mitogen activated protein kinase 1; Mitogen-activated protein kinase 1; Mitogen-activated protein kinase 2; Mitogen-activated protein kinase 3; MNK1; p42-MAPK; p44 MAP kinase; p44-ERK1; p44-MAPK; pp42/MAP kinase; PRKM1; PRKM2; PRKM3; protein tyrosine kinase ERK2
Gene Aliases: 9030612K14Rik; AA407128; AU018647; C78273; ERK; ERK-1; ERK-2; ERK1; ERK2; ERT1; ERT2; Esrk1; HS44KDAP; HUMKER1A; Mapk; MAPK1; MAPK2; MAPK3; Mnk1; Mtap2k; p38; p40; p41; p41mapk; p42-MAPK; P42MAPK; p44; p44-ERK1; p44-MAPK; P44ERK1; P44MAPK; PRKM1; PRKM2; PRKM3