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BV421 Mouse Anti-ERK1/2 (pT202/pY204)
BV421 Mouse Anti-ERK1/2 (pT202/pY204)
Flow cytometric analysis of Erk1/2 (pT202/pY204) expression in stimulated human peripheral blood lymphocytes. Whole blood was either left untreated (dashed line histogram) or treated (solid line histogram) with 400 nM Phorbol 12-Myristate 13-Acetate (PMA; Sigma, Cat. No. P8139) for 15 minutes at 37°C. Erythrocytes were lysed and the leucocytes were fixed with 1X BD Phosflow™ Lyse/Fix Buffer (Cat. No. 558049) for 10 minutes at 37°C. The cells were then permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice for 30 minutes and stained with BD Phosflow™ BV421 Mouse Anti-Erk1/2 (pT202/pY204) (Cat No. 562981). The fluorescence histograms were derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry was performed using a BD LSRFortessa™ Cell Analyzer System.
Flow cytometric analysis of Erk1/2 (pT202/pY204) expression in stimulated human peripheral blood lymphocytes. Whole blood was either left untreated (dashed line histogram) or treated (solid line histogram) with 400 nM Phorbol 12-Myristate 13-Acetate (PMA; Sigma, Cat. No. P8139) for 15 minutes at 37°C. Erythrocytes were lysed and the leucocytes were fixed with 1X BD Phosflow™ Lyse/Fix Buffer (Cat. No. 558049) for 10 minutes at 37°C. The cells were then permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice for 30 minutes and stained with BD Phosflow™ BV421 Mouse Anti-Erk1/2 (pT202/pY204) (Cat No. 562981). The fluorescence histograms were derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry was performed using a BD LSRFortessa™ Cell Analyzer System.
Product Details
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BD Phosflow™
p44/42 MAPK; Extracellular signal-Regulated Kinase 1/2 (pT202/Y204)
Human (QC Testing), Mouse,Rat (Tested in Development)
Mouse IgG1
Phosphorylated Rat ERK1 (T202/Y204) Peptide
Intracellular staining (flow cytometry) (Routinely Tested)
5 µl
AB_2737930
Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
RUO


Preparation And Storage

Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated with BD Horizon BV421 under optimum conditions, and unconjugated antibody and free BD Horizon BV421 were removed.

Product Notices

  1. This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
  2. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  3. An isotype control should be used at the same concentration as the antibody of interest.
  4. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  5. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
  6. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  7. Pacific Blue™ is a trademark of Molecular Probes, Inc., Eugene, OR.
  8. Brilliant Violet™ 421 is a trademark of Sirigen.
  9. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
562981 Rev. 1
Antibody Details
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20A

The members of the Mitogen-Activated Protein Kinase (MAPK) family are components of a key signal transduction cascade that links events at the cell surface to responses in the nucleus. The signaling cascade is found in species as varied as yeast and humans, with many of the proteins being well conserved. In mammals the most widely studied members of the cascade are the Extracellular signal-Regulated Kinases, ERK1 (p44 MAPK) and ERK2 (p42 MAPK).  ERK1 and ERK2 share 85% homology and are activated by extracellular signals such as growth factors, hormones, and phorbol esters. Activation occurs through a series of phosphorylations by kinases activating other kinases and eventually leading to phosphorylation of the ERKs. Growth factor stimulation leads to activation of Ras and Raf, leading to phosphorylation of MEK1 (MAPK/ERK kinase) which, in turn, activates the ERKs via dual phosphorylation. Once activated, the ERKs phosphorylate other cytoplasmic signalling molecules, cell-surface receptors, microtubule-associated proteins, and transcription factors in the nucleus. Thus, the active ERK has myriad downstream effectors that implicate it in the control of cell proliferation and differentiation, as well as regulation of the cytoskeleton. Furthermore, studies have shown that elevated ERK activity is associated with some cancers.

The 20A monoclonal antibody recognizes the phosphorylated threonine 202 and tyrosine 204 (pT202/pY204) of human ERK1 and pT184/pY186 of human ERK2. The orthologous phosphorylation sites in murine ERK1 and ERK2 are T203/Y205 and T183/Y185.

The antibody was conjugated to BD Horizon™ BV421 which is part of the BD Horizon™ Brilliant Violet™ family of dyes.  With an Ex Max of 407-nm and Em Max at 421-nm, BD Horizon™ BV421  can be excited by the violet laser and detected in the standard Pacific Blue™ filter set (eg, 450/50-nm filter). BD Horizon™ BV421  conjugates are very bright, often exhibiting a 10 fold improvement in brightness compared to Pacific Blue™ conjugates.

562981 Rev. 1
Format Details
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BV421
The BD Horizon Brilliant Violet™ 421 (BV421) Dye is part of the BD Horizon Brilliant Violet™ family of dyes. This polymer-technology based dye has an excitation maximum (Ex Max) of 407-nm and an emission maximum (Em Max) at 423-nm. Driven by BD innovation, BV421 is designed to be excited by the violet laser (405-nm) and detected using an optical filter centered near 420-nm (e.g., a 431/28-nm or 450/50-nm bandpass filter). BV421 is an ideal alternative for V450 as it is approximately ten times brighter with less spillover into the BV510/V500 detector. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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BV421
Violet 405 nm
407 nm
423 nm
562981 Rev.1
Citations & References
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Development References (7)

  1. Boulton TG, Cobb MH. Identification of multiple extracellular signal-regulated kinases (ERKs) with antipeptide antibodies. Cell Regul. 1991; 2(5):357-371. (Biology). View Reference
  2. Clark EA, Hynes RO. Ras activation is necessary for integrin-mediated activation of extracellular signal-regulated kinase 2 and cytosolic phospholipase A2 but not for cytoskeletal organization. J Biol Chem. 1996; 271(25):14814-14818. (Biology). View Reference
  3. Irish JM, Czerwinski DK, Nolan GP, Levy R. Altered B-cell receptor signaling kinetics distinguish human follicular lymphoma B cells from tumor-infiltrating nonmalignant B cells. Blood. 2006; 108(9):3135-3142. (Clone-specific: Flow cytometry). View Reference
  4. Irish JM, Czerwinski DK, Nolan GP, Levy R. Kinetics of B cell receptor signaling in human B cell subsets mapped by phosphospecific flow cytometry. J Immunol. 2006; 177(3):1581-1589. (Clone-specific: Flow cytometry). View Reference
  5. Krutzik PO, Nolan GP. Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling. Nat Methods. 2006; 3(5):361-368. (Clone-specific: Flow cytometry). View Reference
  6. Krutzik PO, Nolan GP. Intracellular phospho-protein staining techniques for flow cytometry: monitoring single cell signaling events. Cytometry A. 2003; 55(2):61-70. (Clone-specific: Flow cytometry). View Reference
  7. Sivaraman VS, Wang H, Nuovo GJ, Malbon CC. Hyperexpression of mitogen-activated protein kinase in human breast cancer. J Clin Invest. 1997; 99(7):1478-1483. (Biology). View Reference
View All (7) View Less
562981 Rev. 1

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