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Alexa Fluor® 488 Mouse anti-Elk-1 (pS383)
Alexa Fluor® 488 Mouse anti-Elk-1 (pS383)

Analysis Elk-1 (pS383) in activated human peripheral blood mononuclear cells (PBMC). PBMC were isolated by density gradient centrifugation (Ficoll-Paque™ PLUS, Cat. No. 17-1440-02), and cultured with 20 µg/ml PHA-P (Sigma-Aldrich, Cat. No. L1668) for 3 days, then either left untreated (open histogram) or treated with PMA at

50 nM/10^6 cells for 15 minutes (shaded histogram; Sigma-Aldrich, Cat. No. P8139). Cells were then fixed in BD Cytofix™ buffer (Cat. No. 554655) at 37°C for 10 minutes, then permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice for at least 30 minutes, and then stained with Alexa Fluor® 488 Mouse anti-Elk-1 (pS383). Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

Alexa Fluor® 488 Mouse anti-Elk-1 (pS383)

Western blot analysis of Elk-1 (pS383).  The specificity of mAb M21-1721 was confirmed by western blot analysis using unconjugated Mouse anti-Elk-1 (pS383) antibody on lysates from PBMC that were cultured with PHA for 3 days (lane 1) or PBMC that were cultured with PHA for 3 days and then treated with PMA for 15 minutes (lane 2). Elk-1 (pS383) is identified as a band of 62 kDa in the PMA-treated cells.

Analysis Elk-1 (pS383) in activated human peripheral blood mononuclear cells (PBMC). PBMC were isolated by density gradient centrifugation (Ficoll-Paque™ PLUS, Cat. No. 17-1440-02), and cultured with 20 µg/ml PHA-P (Sigma-Aldrich, Cat. No. L1668) for 3 days, then either left untreated (open histogram) or treated with PMA at

50 nM/10^6 cells for 15 minutes (shaded histogram; Sigma-Aldrich, Cat. No. P8139). Cells were then fixed in BD Cytofix™ buffer (Cat. No. 554655) at 37°C for 10 minutes, then permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice for at least 30 minutes, and then stained with Alexa Fluor® 488 Mouse anti-Elk-1 (pS383). Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

Western blot analysis of Elk-1 (pS383).  The specificity of mAb M21-1721 was confirmed by western blot analysis using unconjugated Mouse anti-Elk-1 (pS383) antibody on lysates from PBMC that were cultured with PHA for 3 days (lane 1) or PBMC that were cultured with PHA for 3 days and then treated with PMA for 15 minutes (lane 2). Elk-1 (pS383) is identified as a band of 62 kDa in the PMA-treated cells.

Product Details
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BD Phosflow™
ELK1
Human (QC Testing), Mouse, Rat (Predicted)
Mouse BALB/c IgG1, κ
Phosphorylated Human Elk-1 Peptide
Intracellular staining (flow cytometry) (Routinely Tested)
20 µl
AB_1645348
Aqueous buffered solution containing BSA, protein stabilizer, 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® 488 under optimum conditions, and unreacted Alexa Fluor® 488 was removed.

Recommended Assay Procedures

This antibody conjugate is suitable for intracellular staining of human peripheral blood mononuclear cells using BD Cytofix™ Fixation Buffer.  Any of the three BD Phosflow™ permeabilization buffers may be used.

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. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  3. Alexa Fluor® 488 fluorochrome emission is collected at the same instrument settings as for fluorescein isothiocyanate (FITC).
  4. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  5. Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
  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. 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.
  8. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  9. Ficoll-Paque is a trademark of Amersham Biosciences Limited.
  10. All other brands are trademarks of their respective owners.
560417 Rev. 2
Antibody Details
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M21-1721

Elk-1 is a 62-kDa member of the Ets oncogene family of transcription factors and a member of the subfamily of ternary complex factors (TCF). Ets proteins mediate a variety of gene activities in response to serum and growth factors. Proteins in the TCF subfamily form a ternary complex by binding to the Serum Response Element (SRE) in conjunction with a dimer of Serum Response Factors (SRF). Elk-1 is phosphorylated by mitogen-activated protein (MAP) kinase pathways in vivo at a cluster of S/T motifs at its carboxy-terminus. Phosphorylation at these sites, particularly at serine 383 (S383), is critical for Elk-1 transcriptional activation. Studies have shown that Elk-1 is a direct target of activated MAP kinase, and that it is also a target of the stress-activated kinase SAPK/JNK.

The M21-1721 monoclonal antibody recognizes the phosphorylated S383 of activated Elk-1. The orthologous phosphorylation sites of mouse and rat Elk-1 are S384 and S382, respectively.

560417 Rev. 2
Format Details
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Alexa Fluor™ 488
Alexa Fluor™ 488 Dye is part of the BD blue family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 494-nm and an emission maximum (Em Max) at 517-nm. Alexa Fluor™ 488 is designed to be excited by the Blue laser (488 nm) and detected using an optical filter centered near 520-nm (e.g., a 530/30-nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
Alexa Fluor™ 488
Blue 488 nm
494 nm
517 nm
560417 Rev.2
Citations & References
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Development References (5)

  1. Bardwell AJ, Abdollahi M, Bardwell L. Docking sites on mitogen-activated protein kinase (MAPK) kinases, MAPK phosphatases and the Elk-1 transcription factor compete for MAPK binding and are crucial for enzymic activity. Biochem J. 2003; 370:1077-1085. (Biology). View Reference
  2. Cavigelli M, Dolfi F, Claret FX, Karin M. Induction of c-fos expression through JNK-mediated TCF/Elk-1 phosphorylation. EMBO J. 1995; 14(23):5957-5964. (Biology). View Reference
  3. Espanel X, Wälchli S, Rückle T et al. Mapping of synergistic components of weakly interacting protein-protein motifs using arrays of paired peptides. J Biol Chem. 2003; 278(17):15162-15167. (Biology: Bioassay). View Reference
  4. Sugimoto T, Stewart S, Guan KL. The calcium/calmodulin-dependent protein phosphatase calcineurin is the major Elk-1 phosphatase. J Biol Chem. 1997; 272(47):29415-29418. (Biology). View Reference
  5. Tian J, Karin M. Stimulation of Elk1 transcriptional activity by mitogen-activated protein kinases is negatively regulated by protein phosphatase 2B (calcineurin). J Biol Chem. 1999; 274(21):15173-15180. (Biology). View Reference
View All (5) View Less
560417 Rev. 2

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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.