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PE Mouse anti-MEK1
PE Mouse anti-MEK1

Analysis of MEK1 in HeLaS3 cells.  HeLaS3 cells were either transfected with MEK1 RNAi (open histogram) or untreated (shaded histogram).  The cells were fixed (BD Cytofix™ Fixation buffer, Cat. No. 554655) for 10 minutes at 37°C, then permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for 30 minutes, and then stained with PE Mouse anti-MEK1 (Cat. No. 560099).  Down-regulation of MEK1 expression is evident in the RNAi-transfected cells.  Flow cytometry was performed on a BD™ LSR II flow cytometry system.

Analysis of MEK1 in HeLaS3 cells.  HeLaS3 cells were either transfected with MEK1 RNAi (open histogram) or untreated (shaded histogram).  The cells were fixed (BD Cytofix™ Fixation buffer, Cat. No. 554655) for 10 minutes at 37°C, then permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for 30 minutes, and then stained with PE Mouse anti-MEK1 (Cat. No. 560099).  Down-regulation of MEK1 expression is evident in the RNAi-transfected cells.  Flow cytometry was performed on a BD™ LSR II flow cytometry system.

Product Details
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BD Phosflow™
MAPK/ERK kinase 1, EC 2.7.12.2, kinase MEK1, MAPKK1, PRKMK1
Human (QC Testing), Mouse, Rat, Dog, Chicken (Reported)
Mouse IgG2a
Human MEK1 Recombinant Protein
Intracellular staining (flow cytometry) (Routinely Tested)
20 µl
AB_1645608
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 R-PE under optimum conditions, and unconjugated antibody and free PE were removed.

Recommended Assay Procedures

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

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. An isotype control should be used at the same concentration as the antibody of interest.
  3. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  4. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  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. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
560099 Rev. 2
Antibody Details
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25/MEK1

MEK1 (MapK/ERK Kinase 1) is a 45-kDa member of the MEK family of dual specificity kinases.  MEK is activated by a variety of cellular serine/threonine kinases including c-Raf, A-Raf, c-mos, and MEK Kinase-1.  Activated MEK phosphorylates MAP kinase (ERK) at threonine and tyrosine residues.  This results in activation of ERK and its signaling pathway. MEK is highly specific for ERK and various MEKs preferentially phosphorylate individual ERK isoforms.  MEK1 only activates ERK1 and ERK2.  This specificity may result from variations in ERK regions that are known as the phosphorylation lip and kinase backbone.  MEK's localization is cytoplasmic, but mitogenic stimulation induces a mass translocation to the nucleus.  Mechanisms behind this nuclear translocation remain unknown.  However, MEK contains an N-terminal nuclear export signal (NES) that mediates its rapid exodus from the nucleus and restores its unstimulated cellular distribution.

The 25/MEK1 monoclonal antibody recognizes MEK1, regardless of phosphorylation status.

The specificity of this antibody conjugate for flow cytometric analysis was validated by confirming that RNA-mediated interference (RNAi) of the specific protein reduced the staining of the cells (see figure). Furthermore, the capacity of the RNAi to down-regulate the expression of the relevant protein was confirmed by western blot analysis.

560099 Rev. 2
Format Details
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PE
R-Phycoerythrin (PE), is part of the BD family of Phycobiliprotein dyes. This fluorochrome is a multimeric fluorescent phycobiliprotein with excitation maximum (Ex Max) of 496 nm and 566 nm and an emission maximum (Em Max) at 576 nm. PE is designed to be excited by the Blue (488 nm), Green (532 nm) and Yellow-Green (561 nm) lasers and detected using an optical filter centered near 575 nm (e.g., a 575/26-nm bandpass filter). As PE is excited by multiple lasers, this can result in cross-laser excitation and fluorescence spillover on instruments with various combinations of Blue, Green, and Yellow-Green lasers. Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
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PE
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
576 nm
560099 Rev.2
Citations & References
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Development References (4)

  1. Aplin AE, Stewart SA, Assoian RK, Juliano RL. Integrin-mediated adhesion regulates ERK nuclear translocation and phosphorylation of Elk-1. J Cell Biol. 2001; 153(2):273-282. (Biology: Immunofluorescence, Western blot). View Reference
  2. Gu J, Fujibayashi A, Yamada KM, Sekiguchi K. Laminin-10/11 and fibronectin differentially prevent apoptosis induced by serum removal via phosphatidylinositol 3-kinase/Akt- and MEK1/ERK-dependent pathways. J Biol Chem. 2002; 277(22):19922-19928. (Clone-specific: Western blot). View Reference
  3. Robinson MJ, Cheng M, Khokhlatchev A, et al. Contributions of the mitogen-activated protein (MAP) kinase backbone and phosphorylation loop to MEK specificity. J Biol Chem. 1996; 271(47):29734-29739. (Biology). View Reference
  4. Short SM, Boyer JL, Juliano RL. Integrins regulate the linkage between upstream and downstream events in G protein-coupled receptor signaling to mitogen-activated protein kinase. J Biol Chem. 2000; 275(17):12970-12977. (Clone-specific: Immunoprecipitation, In vitro kinase assay, Western blot). View Reference
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560099 Rev. 2

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