Immunoprecipitation of α-Parvin from HeLa cell extracts, using Rabbit (DA1E) mAb IgG XP® Isotype Control (Sepharose® Bead Conjugate) #3423 (Lane 1) or α-Parvin (D7F9) XP® Rabbit mAb (Sepharose® Bead Conjugate) (Lane 2). Lane 3 is 10% input. Western blot analysis was performed using α-Parvin (D7F9) XP® Rabbit mAb #8190 and Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127.
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This Cell Signaling Technology antibody is immobilized via covalent binding of primary amino groups to N-hydroxysuccinimide (NHS)-activated Sepharose® beads. α-Parvin (D7F9) XP® Rabbit mAb (Sepharose® Bead Conjugate) is useful for the immunoprecipitation of α-Parvin. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated α-Parvin (D7F9) XP® Rabbit mAb #8190.
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol. Store at –20°C. Do not aliquot the antibodies.
This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
10X Cell Lysis Buffer: (#9803) 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml Leupeptin
NOTE: CST recommends adding 1 mM PMSF (#8553) before use*.
Proceed to one of the following specific set of steps.
NOTE: To minimize masking caused by denatured IgG heavy chains (~50 kDa), we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (L57A3) mAb (#3677) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127). To minimize masking caused by denatured IgG light chains (~25 kDa), we recommend using Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127).
posted December 2007
Protocol Id: 27
α-Parvin (D7F9) XP® Rabbit mAb (Sepharose® Bead Conjugate) recognizes endogenous levels of total α-parvin protein.Species Reactivity:
Human, Mouse, Rat, Monkey, Dog
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human α-parvin protein.
The extracellular matrix (ECM) is a complex structure of secreted macromolecules surrounding mammalian organs and tissues. Controlled interactions between cells and the ECM are important in proliferation, migration, survival, polarity, and differentiation. Cells contact the ECM primarily through heterodimeric integral membrane proteins called integrins. Integrins connect the ECM to the cytoskeleton, and therefore the cell signaling machinery, through protein complexes called focal adhesions (1).
The ILK/PINCH/Parvin (IPP) complex is composed of three highly conserved proteins recruited to sites of ECM contact as pre-assembled structures. The IPP acts at the interface of the integrin/actin connection to regulate formation of focal adhesions and integrin signaling. All three proteins contain multiple protein binding domains allowing them to function as adaptor proteins in the formation of focal adhesions. ILK (integrin-linked kinase) also has a catalytic (protein Ser/Thr kinase) domain, and may or may not function as a kinase in vivo. Roles for IPP proteins outside of the IPP complex have been proposed, including regulation of gene expression (2,3).
The parvin family consists of 3 members, α-parvin/actopaxin, β-parvin/affixin, and γ-parvin. α-parvin and β-parvin are expressed ubiquitously, while expression of γ-parvin is restricted to hematopoietic cells (4). α-parvin binds to f-actin both directly and via interaction with the focal adhesion protein paxillin (5). α-parvin regulates cell spreading and motility through interactions with the cofilin kinase TESK1 (6), and with the GTPase activating protein CdGAP (7). Phosphorylation of α-parvin during mitosis may have a role in the regulation of actin dynamics during the cell cycle (8).
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