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    Alexa Fluor® 647 Mouse anti-MEK1 (pS298)
    Alexa Fluor® 647 Mouse anti-MEK1 (pS298)
    Analysis of MEK1 (pS298) in human epithelioid carcinoma.  Hela S3 cells (ATCC CCL 2.2) were serum starved overnight, detached using 1X trypsin, washed, resuspended in serum-free DMEM, and then either left unstimulated (open histogram) or stimulated (shaded histogram) with 50 nM Calyculin A (Calbiochem, Cat. No. 208851) at 37˚C for 30 minutes.  The cells were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes at 37°C, then permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for at least 30 minutes, and then stained with Alexa Fluor® 647 Mouse anti-MEK1 (pS298).  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.
    Alexa Fluor® 647 Mouse anti-MEK1 (pS298)
    Analysis of MEK1 (pS298) in human epithelioid carcinoma.  Hela S3 cells (ATCC CCL 2.2) were serum starved overnight, detached using 1X trypsin, washed, resuspended in serum-free DMEM, and then either left unstimulated (open histogram) or stimulated (shaded histogram) with 50 nM Calyculin A (Calbiochem, Cat. No. 208851) at 37˚C for 30 minutes.  The cells were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes at 37°C, then permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for at least 30 minutes, and then stained with Alexa Fluor® 647 Mouse anti-MEK1 (pS298).  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.
    Product Details
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    BD Phosflow™
    MAP2K1, MAPKK1, MKK1, MP2K1, PPKMK1
    Human (QC Testing), Mouse (Tested in Development)
    Mouse BALB/c IgG1, κ
    Phosphorylated Human MEK1 Peptide
    Intracellular staining (flow cytometry) (Routinely Tested)
    20 µl
    AB_1645425
    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 to Alexa Fluor® 647 under optimum conditions, and unreacted Alexa Fluor® 647 was removed.
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    Recommended Assay Procedures

    Either BD Cytofix™ fixation buffer or BD Phosflow™ Fix Buffer I may be used for cell fixation.

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    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. 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.
    4. Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
    5. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
    6. The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
    7. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
    8. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
    9. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
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    560043 Rev. 2
    Antibody Details
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    J114-64

    MEK (Map/Erk Kinase) 1 and 2 are serine/threonine kinases, also known as MAP kinase kinases (MAP2K1 and 2, MAPKK1 and 2, or MKK1 and 2).  They activate the MAP (Mitogen-Activated Protein) kinases, also known as ERKs (Extracellular signal Regulated Kinases), which are critical kinases in multiple signal transduction pathways that regulate cell growth and differentiation.  Activation of MEK 1 and 2 is dependent upon phosphorylation of serines 218 and/or 222 by activated MAP kinase kinase kinases (MAP3Ks), such as the Raf isoforms.  Hormones, growth and differentiating factors, or tumor promoters induce Raf activation via activation of Ras proteins.  Alternatively, cellular adhesion can lead to phosphorylation of MEK1 at serine 298 (S298), mediated by p21-activated kinase (PAK).  The S298-phosphorylated MEK1 has an enhanced capacity to interact with Raf, resulting in MEK1 activation.

    The J114-64 monoclonal antibody recognizes the phosphorylated S298 of MEK1.

    560043 Rev. 2
    Format Details
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    Alexa Fluor™ 647
    Alexa Fluor™ 647 Dye is part of the BD red family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 653-nm and an emission maximum (Em Max) at 669-nm. Alexa Fluor 647 is designed to be excited by the Red laser (627-640 nm) and detected using an optical filter centered near 520-nm (e.g., a 660/20 nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
    Alexa Fluor™ 647
    Red 627-640 nm
    653 nm
    669 nm
    560043 Rev.2
    Citations & References
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    Development References (3)

    1. Eblen ST, Slack JK, Weber MJ, Catling AD. Rac-PAK signaling stimulates extracellular signal-regulated kinase (ERK) activation by regulating formation of MEK1-ERK complexes. Mol Cell Biol. 2002; 22(17):6023-6033. (Biology).
    2. Kolch W. Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions. Biochem J. 2000; 351:289-305. (Biology). View Reference
    3. Slack-Davis JK, Eblen ST, Zecevic M, et al. PAK1 phosphorylation of MEK1 regulates fibronectin-stimulated MAPK activation. J Cell Biol. 2003; 162(2):281-291. (Biology).
    560043 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.

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