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PE Mouse anti-Elk-1 (pS383)
PE 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™ Fixation 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 PE Mouse anti-Elk-1 (pS383). Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.

PE 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™ Fixation 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 PE 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_1645505
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

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. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
  4. Ficoll-Paque is a trademark of Amersham Biosciences Limited.
  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. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
560412 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.

560412 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.
altImg
PE
Yellow-Green 488 nm, 532 nm, 561 nm
496 nm, 566 nm
576 nm
560412 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). 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
560412 Rev. 2

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