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BD Phosflow™ PE Mouse anti-SLP-76 (pY128)
Clone J141-668.36.58 (RUO)
Analysis of SLP-76 (pY128) in human peripheral blood lymphocytes. Human peripheral blood mononuclear cells were either stimulated by cross-linking of CD3 and CD28 with NA/LE Mouse anti-Human CD3 (Cat. No. 555329) and NA/LE Mouse anti-Human CD28 (Cat. No. 555725) on ice for 15 minutes, followed by Purified Goat anti-Mouse Ig (Cat. No. 553998) on ice for 15 minutes, then allowed to undergo phosphorylation at 37°C for 5 minutes (shaded histograms) or unstimulated (open histograms). The cells were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes at 37°C, permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for 30 minutes, blocked with normal mouse immunoglobulin, and then stained with PE Mouse anti-SLP-76 (pY128, Cat. No. 558437) and PerCP-Cy5.5 Mouse anti-human CD3 (Cat. No. 340949). The left panel displays the upregulated phosphorylation of SLP-76 in activated CD3-positive T lymphocytes, while the right panel displays the lack of upregulated SLP-76 phosphorylation in CD3-negative non-T cells. Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.
Analysis of SLP-76 (pY128) in human peripheral blood lymphocytes. Human peripheral blood mononuclear cells were either stimulated by cross-linking of CD3 and CD28 with NA/LE Mouse anti-Human CD3 (Cat. No. 555329) and NA/LE Mouse anti-Human CD28 (Cat. No. 555725) on ice for 15 minutes, followed by Purified Goat anti-Mouse Ig (Cat. No. 553998) on ice for 15 minutes, then allowed to undergo phosphorylation at 37°C for 5 minutes (shaded histograms) or unstimulated (open histograms). The cells were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes at 37°C, permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for 30 minutes, blocked with normal mouse immunoglobulin, and then stained with PE Mouse anti-SLP-76 (pY128, Cat. No. 558437) and PerCP-Cy5.5 Mouse anti-human CD3 (Cat. No. 340949). The left panel displays the upregulated phosphorylation of SLP-76 in activated CD3-positive T lymphocytes, while the right panel displays the lack of upregulated SLP-76 phosphorylation in CD3-negative non-T cells. Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.
Analysis of SLP-76 (pY128) in human peripheral blood lymphocytes. Human peripheral blood mononuclear cells were either stimulated by cross-linking of CD3 and CD28 with NA/LE Mouse anti-Human CD3 (Cat. No. 555329) and NA/LE Mouse anti-Human CD28 (Cat. No. 555725) on ice for 15 minutes, followed by Purified Goat anti-Mouse Ig (Cat. No. 553998) on ice for 15 minutes, then allowed to undergo phosphorylation at 37°C for 5 minutes (shaded histograms) or unstimulated (open histograms). The cells were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes at 37°C, permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for 30 minutes, blocked with normal mouse immunoglobulin, and then stained with PE Mouse anti-SLP-76 (pY128, Cat. No. 558437) and PerCP-Cy5.5 Mouse anti-human CD3 (Cat. No. 340949). The left panel displays the upregulated phosphorylation of SLP-76 in activated CD3-positive T lymphocytes, while the right panel displays the lack of upregulated SLP-76 phosphorylation in CD3-negative non-T cells. Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.
Analysis of SLP-76 (pY128) in human peripheral blood lymphocytes. Human peripheral blood mononuclear cells were either stimulated by cross-linking of CD3 and CD28 with NA/LE Mouse anti-Human CD3 (Cat. No. 555329) and NA/LE Mouse anti-Human CD28 (Cat. No. 555725) on ice for 15 minutes, followed by Purified Goat anti-Mouse Ig (Cat. No. 553998) on ice for 15 minutes, then allowed to undergo phosphorylation at 37°C for 5 minutes (shaded histograms) or unstimulated (open histograms). The cells were fixed (BD Cytofix™ buffer, Cat. No. 554655) for 10 minutes at 37°C, permeabilized (BD Phosflow™ Perm Buffer III, Cat. No. 558050) on ice for 30 minutes, blocked with normal mouse immunoglobulin, and then stained with PE Mouse anti-SLP-76 (pY128, Cat. No. 558437) and PerCP-Cy5.5 Mouse anti-human CD3 (Cat. No. 340949). The left panel displays the upregulated phosphorylation of SLP-76 in activated CD3-positive T lymphocytes, while the right panel displays the lack of upregulated SLP-76 phosphorylation in CD3-negative non-T cells. Flow cytometry was performed on a BD FACSCalibur™ flow cytometry system.
Product Details
BD Phosflow™
Human, Mouse (QC Testing), Rat (Predicted)
Mouse BALB/c IgG1, κ
Phosphorylated Human SLP-76
Intracellular staining (flow cytometry) (Routinely Tested)
20 µl
AB_647246
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 antibody was conjugated with R-PE under optimum conditions, and unconjugated antibody and free PE were removed. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.
Recommended Assay Procedures
This antibody conjugate is suitable for intracellular staining of human whole blood and mouse splenocytes (using BD Phosflow™ Lyse/Fix Buffer) and peripheral blood mononuclear cells (using BD Cytofix™ Fixation Buffer). Any of the three BD Phosflow™ permeabilization buffers may be used.
Product Notices
- 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.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Cy is a trademark of GE Healthcare.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
Stain Buffer (FBS) RUO
Size
500 ML
Cat No.
554656
Stain Buffer (BSA) RUO
Size
500 ML
Cat No.
554657
Lyse/Fix Buffer 5X RUO
Size
250 ML
Cat No.
558049
Perm Buffer III RUO
Size
125 ML
Cat No.
558050
Perm Buffer II RUO
Size
125 ML
Cat No.
558052
Perm/Wash Buffer I RUO
Size
125 ML
Cat No.
557885
558437 Rev. 2
Antibody Details
J141-668.36.58
SLP-76 (SH2 domain-containing Leukocyte Protein of 76 kDa) is a tyrosine phosphoprotein that is involved in the T cell receptor (TCR)-mediated intracellular signaling pathway. It may be involved in the signaling pathways of other peripheral blood leukocytes; thymic/splenic cells; and in human T, B, and monocytic cell lines. SLP-76 consists of several motifs that signify its importance in protein-protein interactions involved in intracellular signaling pathways, such as the SH2 domain in the C-terminus, the three amino-terminus 17-amino acid repeats with conserved tyrosine and acidic residues (DYE(S/P)P), and a proline rich region. SLP-76 has been shown to associate with Gads, Grb2, PLCγ1, SLAP-130, and Vav, all of which are part of the signaling cascade in T lymphocytes. An early event in the T cell activation pathway is the phosphorylation, by the Syk-family kinase ZAP-70, of SLP-76 at the three conserved tyrosine motifs, which then mediate interactions with downstream effectors. The phosphorylated tyrosine 128 (Y128) brings the Rho family guanine nucleotide exchange factor Vav1 and the Nck adapter protein, which binds to p21-activated kinase (PAK1) and Wiskott-Aldrich syndrome protein (WASP), into the activation complex. Vav1, PAK1, and WASP may mediate TCR-stimulated actin cytoskeletal rearrangement.
The J141-688.36.58 monoclonal antibody recognizes the phosphorylated Y128 of activated SLP-76.
558437 Rev. 2
Format Details
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.
PE
496 nm, 566 nm
576 nm
558437 Rev.2
Citations & References
View product citations for antibody "558437" on CiteAb
Development References (3)
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Fang N, Motto DG, Ross SE, Koretzky GA. Tyrosines 113, 128, and 145 of SLP-76 are required for optimal augmentation of NFAT promoter activity. J Immunol. 1996; 157:3769-3773. (Biology). View Reference
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Janssen E, Zhang W. Adaptor proteins in lymphocyte activation. Curr Opin Immunol. 2003; 15:269-276. (Biology). View Reference
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Wu JN, Koretzky GA. The SLP-76 family of adapter proteins. Semin Immunol. 2004; 16:379-393. (Biology). View Reference
558437 Rev. 2
Please refer to Support Documents for Quality Certificates
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.