Analysis of WIP (pS488) in human T leukemia cells. LEFT: The shaded histogram displays Jurkat cells (ATCC TIB152) that were stimulated by cross-linking of CD3 and CD28 with NA/LE Mouse anti-Human CD3 mAb (Cat. No. 555329) and NA/LE Mouse anti-Human CD28 mAb (Cat. No. 555725) on ice for 15 minutes followed by Purified Goat anti-Mouse Ig (Cat. No. 553998) on ice for 15 minutes, and then allowed to undergo phosphorylation at 37°C for 3 minutes. The open histogram shows unstimulated Jurkat cells. The cells were fixed using BD Cytofix™ buffer (Cat. No. 554655) for 10 minutes at 37°C, permeabilized usingBD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice for at least 30 minutes, blocked with normal mouse immunoglobulin, and then stained with Alexa Fluor® 647 Mouse anti-WIP (pS488). Flow cytometry was performed on a BD FACSArray™ bioanalyzer system. RIGHT: The specificity of mAb K32-824 was confirmed by western blot using unconjugated antibody on lysates from control (left panel) and CD3/CD28-cross-linked (right panel) Jurkat cells. WIP (pS488) is upregulated in the treated cells; its observed molecular weight is ~60 kDa, although the calculated molecular weight is 51 kDa.
Analysis of WIP (pS488) in human T leukemia cells. LEFT: The shaded histogram displays Jurkat cells (ATCC TIB152) that were stimulated by cross-linking of CD3 and CD28 with NA/LE Mouse anti-Human CD3 mAb (Cat. No. 555329) and NA/LE Mouse anti-Human CD28 mAb (Cat. No. 555725) on ice for 15 minutes followed by Purified Goat anti-Mouse Ig (Cat. No. 553998) on ice for 15 minutes, and then allowed to undergo phosphorylation at 37°C for 3 minutes. The open histogram shows unstimulated Jurkat cells. The cells were fixed using BD Cytofix™ buffer (Cat. No. 554655) for 10 minutes at 37°C, permeabilized usingBD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice for at least 30 minutes, blocked with normal mouse immunoglobulin, and then stained with Alexa Fluor® 647 Mouse anti-WIP (pS488). Flow cytometry was performed on a BD FACSArray™ bioanalyzer system. RIGHT: The specificity of mAb K32-824 was confirmed by western blot using unconjugated antibody on lysates from control (left panel) and CD3/CD28-cross-linked (right panel) Jurkat cells. WIP (pS488) is upregulated in the treated cells; its observed molecular weight is ~60 kDa, although the calculated molecular weight is 51 kDa.
Product Details
BD Phosflow™
PRPL-2 protein, WAIP, WASIP, WASPIP
Human (QC Testing), Mouse,Rat (Predicted)
Mouse IgG1, κ
Phosphorylated Human WIP Peptide
Intracellular staining (flow cytometry) (Routinely Tested)
20 µl
AB_1645450
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 to Alexa Fluor® 647 under optimum conditions, and unreacted Alexa Fluor® 647 was removed.
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).
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
- Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
- 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.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- 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.
Companion Products
Fixation Buffer RUO
Size
100 mL
Cat No.
554655
Perm Buffer III RUO
Size
125 mL
Cat No.
558050
558674 Rev. 2
Antibody Details
K32-824
Wiskott-Adrich syndrome protein (WASP)-Interacting Protein (WIP) is a member of the verprolin family of proteins that regulate cytoskeletal organization in a wide variety of cellular activities, including endocytosis, cellular adhesion and migration, mast cell degranulation, and lymphocyte activation. The 503-amino acid WIP protein contains binding sites for actin (globular and filamentous) and other proteins that are involved in the regulation of actin polymerization, such as WASP, N-WASP, profilin, cortactin, Hck, and NCK. As its functions imply, WIP is localized in actin-rich cell structures.
The K32-824 monoclonal antibody recognizes the phosphorylated serine 488 (pS488) of human WIP. The orthologous phosphorylation sites in mouse and rat WIP are S478 and S472, respectively.
558674 Rev. 2
Format Details
Alexa Fluor™ 647
Alexa Fluor™ 647 Dye is part of the BD red family of dyes. This is a small organic fluorochrome with an excitation maximum (Ex Max) at 653-nm and an emission maximum (Em Max) at 669-nm. Alexa Fluor™ 647 is designed to be excited by the Red laser (627-640 nm) and detected using an optical filter centered near 670-nm (e.g., a 660/20 nm bandpass filter). Please ensure that your instrument’s configurations (lasers and optical filters) are appropriate for this dye.
Alexa Fluor™ 647
Red 627-640 nm
653 nm
669 nm
558674 Rev.2
Citations & References
View product citations for antibody "558674" on CiteAb
Development References (3)
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Antón IM, Jones GE. WIP: A multifunctional protein involved in actin cytoskeleton regulation. Eur J Cell Biol. 2006; 85:295-304. (Biology).
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Aspenström P. The verprolin family of proteins: Regulators of cell morphogenesis and endocytosis. FEBS Lett. 2005; 579:5253-5259. (Biology).
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Sechi AS, Wehland J. Interplay between TCR signalling and actin cytoskeleton dynamics. Trends Immunol. 2004; 25(5):257-265. (Biology).
558674 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.