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PerCP-Cy™5.5 Mouse anti-ERK1/2 (pT202/pY204)
PerCP-Cy™5.5 Mouse anti-ERK1/2 (pT202/pY204)

Analysis of Erk1/2 (pT202/pY204) in human peripheral blood lymphocytes. Whole blood was either left untreated (unshaded) or treated (shaded) with 400nM of phorbol 12-myristate 13-acetate (PMA) (Sigma, Cat.# P8139) for 15 minutes at 37°C. The samples were lysed and fixed with 1X BD Phosflow™ Lyse/Fix buffer (Cat. No. 558049) for 10 minutes at 37°C, permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for 30 minutes and were then stained with PerCP-CY™5.5 anti-Erk1/2 (pT202/pY204). For data analysis, lymphocytes were selected by scatter profile. Flow cytometry was performed on a BD FACSCalibur™ II flow cytometry system.

Analysis of Erk1/2 (pT202/pY204) in human peripheral blood lymphocytes. Whole blood was either left untreated (unshaded) or treated (shaded) with 400nM of phorbol 12-myristate 13-acetate (PMA) (Sigma, Cat.# P8139) for 15 minutes at 37°C. The samples were lysed and fixed with 1X BD Phosflow™ Lyse/Fix buffer (Cat. No. 558049) for 10 minutes at 37°C, permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for 30 minutes and were then stained with PerCP-CY™5.5 anti-Erk1/2 (pT202/pY204). For data analysis, lymphocytes were selected by scatter profile. Flow cytometry was performed on a BD FACSCalibur™ II flow cytometry system.

Product Details
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BD Phosflow™
p44/42 MAPK; Extracellular signal-Regulated Kinase 1/2 (pT202/Y204)
Human (QC Testing), Human, Mouse, Rat (Reactivity Confirmed in Development)
Mouse IgG1
Phosphorylated Rat ERK1 (T202/Y204) Peptide
Intracellular staining (flow cytometry) (Routinely Tested)
20 µl
AB_1645298
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 PerCP-Cy5.5 under optimum conditions, and unconjugated antibody and free PerCP-Cy5.5 were removed. Storage of PerCP-Cy5.5 conjugates in unoptimized diluent is not recommended and may result in loss of signal intensity.

Recommended Assay Procedures

This antibody conjugate is suitable for intracellular staining of human whole blood (using BD™ Phosflow Lyse/Fix Buffer) and peripheral blood mononuclear cells (using BD Cytofix™ Fixation Buffer or BD Phosflow™ Fix Buffer I).  Any of the three BD Phosflow™ permeabilization buffers may be used.

This mAb was characterized by flow cytometry (Flow) and western blot analysis (WB) using these model systems:

Method                        Species                Cells                        Treatment                Fixation                        Perm buffer                Result

Flow                                Human                PBMC                        PMA                        Fixation Buffer        I, II or III                        Positive Staining

Flow                                Human                Whole Blood                PMA                        Lyse/Fix                        I, II or III                        Positive Staining

WB                                Human                A431 Cell Lysate        EGF                                Not Applicable        Not Applicable        44/42 kDa

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. Please observe the following precautions: Absorption of visible light can significantly alter the energy transfer occurring in any tandem fluorochrome conjugate; therefore, we recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to prevent exposure of conjugated reagents, including cells stained with those reagents, to room illumination.
  3. PerCP-Cy5.5–labelled antibodies can be used with FITC- and R-PE–labelled reagents in single-laser flow cytometers with no significant spectral overlap of PerCP-Cy5.5, FITC, and R-PE fluorescence.
  4. PerCP-Cy5.5 is optimized for use with a single argon ion laser emitting 488-nm light. Because of the broad absorption spectrum of the tandem fluorochrome, extra care must be taken when using dual-laser cytometers, which may directly excite both PerCP and Cy5.5™. We recommend the use of cross-beam compensation during data acquisition or software compensation during data analysis.
  5. This product is subject to proprietary rights of Amersham Biosciences Corp. and Carnegie Mellon University and made and sold under license from Amersham Biosciences Corp. This product is licensed for sale only for research. It is not licensed for any other use. If you require a commercial license to use this product and do not have one return this material, unopened to BD Biosciences, 10975 Torreyana Rd, San Diego, CA 92121 and any money paid for the material will be refunded.
  6. Cy is a trademark of Amersham Biosciences Limited. This conjugated product is sold under license to the following patents: US Patent Nos. 5,486,616; 5,569,587; 5,569,766; 5,627,027.
  7. 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.
  8. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  9. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  10. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
560115 Rev. 2
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.

560115 Rev. 2
Format Details
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PerCP-Cy5.5
PerCP-Cy5.5 dye is part of the BD blue family of dyes. This tandem fluorochrome is comprised of a fluorescent protein complex (PerCP) with an excitation maximum (Ex Max) of 482 nm and an acceptor dye with an emission maximum (Em Max) at 676 nm. PerCP-Cy5 is designed to be excited by the blue laser (488-nm) and detected using an optical filter centered near 680 nm (e.g., a 695/40 nm bandpass filter). The donor dye can be partially excited by the Violet (405-nm) laser resulting in cross-laser excitation and fluorescence spillover. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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PerCP-Cy5.5
Blue 488 nm
482 nm
676 nm
560115 Rev.2
Citations & References
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Development References (4)

  1. Cobb MH, Boulton TG, Robbins DJ. Extracellular signal-regulated kinases: ERKs in progress. Cell Regul. 1991; 2:965-978. (Biology). View Reference
  2. Kim SC, Hahn JS, Min YH, Yoo NC, Ko YW, Lee WJ. Constitutive activation of extracellular signal-regulated kinase in human acute leukemias: combined role of activation of MEK, hyperexpression of extracellular signal-regulated kinase, and downregulation of a phosphatase, PAC1. Blood. 1999; 93(11):3893-3899. (Clone-specific: In vitro kinase assay, Western blot). View Reference
  3. 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
  4. Treisman R. Regulation of transcription by MAP kinase cascades. Curr Opin Cell Biol. 1996; 8:205-215. (Biology). View Reference
View All (4) View Less
560115 Rev. 2

Please refer to Support Documents for Quality Certificates


Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described


Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims.  Comparisons are not made against non-BD technologies, unless otherwise noted.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.

Refer to manufacturer's instructions for use and related User Manuals and Technical Data Sheets before using this product as described.

Comparisons, where applicable, are made against older BD technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.