Preparation And Storage
Recommended Assay Procedures
Bioimaging
1. Seed the cells in appropriate culture medium at ~10,000 cells per well in a BD Falcon™ 96-well Imaging Plate (Cat. No. 353219) and culture overnight.
2. Remove the culture medium from the wells, and fix the cells by adding 100 μl of BD Cytofix™ Fixation Buffer (Cat. No. 554655) to each well. Incubate for 10 minutes at room temperature (RT).
3. Remove the fixative from the wells, and permeabilize the cells using either BD Perm Buffer III, 90% methanol, or Triton™ X-100:
a. Add 100 μl of -20°C 90% methanol or Perm Buffer III (Cat. No. 558050) to each well and incubate for 5 minutes at RT.
OR
b. Add 100 μl of 0.1% Triton™ X-100 to each well and incubate for 5 minutes at RT.
4. Remove the permeabilization buffer, and wash the wells twice with 100 μl of 1× PBS.
5. Remove the PBS, and block the cells by adding 100 μl of BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) to each well. Incubate for 30 minutes at RT.
6. Remove the blocking buffer and add 50 μl of the optimally titrated primary antibody (diluted in Stain Buffer) to each well, and incubate for 1 hour at RT.
7. Remove the primary antibody, and wash the wells three times with 100 μl of 1× PBS.
8. Remove the PBS, and add the second step reagent at its optimally titrated concentration in 50 μl to each well, and incubate in the dark for 1 hour at RT.
9. Remove the second step reagent, and wash the wells three times with 100 μl of 1× PBS.
10. Remove the PBS, and counter-stain the nuclei by adding 200 μl per well of 2 μg/ml Hoechst 33342 (e.g., Sigma-Aldrich Cat. No. B2261) in 1× PBS to each well at least 15 minutes before imaging.
11. View and analyze the cells on an appropriate imaging instrument.
Bioimaging: For more detailed information please refer to http://www.bdbiosciences.com/support/resources/protocols/ceritifed_reagents.jsp
Western blot: For more detailed information please refer to http://www.bdbiosciences.com/pharmingen/protocols/Western_Blotting.shtml
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- This antibody has been developed and certified for the bioimaging application. However, a routine bioimaging test is not performed on every lot. Researchers are encouraged to titrate the reagent for optimal performance.
- 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.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- Triton is a trademark of the Dow Chemical Company.
Companion Products
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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.
Development References (5)
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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. (Clone-specific: Fluorescence microscopy, Immunofluorescence, Western blot). View Reference
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Freeman WM, Brebner K, Lynch WJ, et al. Changes in rat frontal cortex gene expression following chronic cocaine. Brain Res Mol Brain Res. 2002; 104(1):11-20. (Clone-specific: Western blot). View Reference
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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
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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
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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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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.
