Alexa Fluor® 647 Mouse anti-MEK1
Clone 25/MEK1 (RUO)
- Brand BD Phosflow™
- Alternative Name MAPK/ERK kinase 1, EC 126.96.36.199, kinase MEK1, MAPKK1, PRKMK1
- Vol. Per Test 20 µl
- Isotype Mouse IgG2a
- Reactivity Human (QC Testing) Mouse, Rat, Dog, Chicken (Reported)
Intracellular staining (flow cytometry) (Routinely Tested)
- Immunogen Human MEK1 Recombinant Protein
- Storage Buffer Aqueous buffered solution containing BSA and ≤0.09% sodium azide.
- Regulatory Status RUO
Regulatory Status Legend
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.
Alexa Fluor® 647 conjugates are highly photostable and remain fluorescent over a broad pH range. The excitation and emission maxima are nearly identical to those of APC. However, APC tends to be brighter while Alexa Fluor® 647 is more optimal for intracellular applications. This fluorochrome exhibits uncommon photostability, making it an ideal choice for use in fluorescence microscopy. Due to nearly identical excitation and emission properties but different spillover characteristics, APC and Alexa Fluor® 647 cannot be used simultaneously.
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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.
- 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).
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- 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.
- Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- 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.
- Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
- Please refer to www.bdbiosciences.com/pharmingen/protocols for technical protocols.
Either BD Cytofix™ fixation buffer or BD Phosflow™ Fix Buffer I may be used for cell fixation.
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 HeLaS3 RNAi BD Cytofix™ III Down-regulation
Flow Human PBMC Not Applicable BD Cytofix™ I, II or III Positive Staining
WB Human A431 Cell Lysate Not Applicable Not Applicable Not Applicable 45 kDa