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PE Mouse anti-MEK1 (pS298)
PE 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 PE Mouse anti-MEK1 (pS298, Cat. No. 560042).  Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

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 PE Mouse anti-MEK1 (pS298, Cat. No. 560042).  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_1645520
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™ 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. 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. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  5. Species cross-reactivity detected in product development may not have been confirmed on every format and/or application.
  6. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
560042 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.

560042 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
560042 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).
560042 Rev. 2

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