Anti-Alix/Xp95 Antibody, clone 1A3

Programmed cell death 6-interacting protein (UniProt Q8WUM4; also known as ALG-2-interacting protein 1, ALG-2-interacting protein X, Apoptosis-linked gene-2-interacting protein X, Hp95, Human ortholog of Xenopus protein of 95 kDa, Human ortholog of Xp95, PDCD6-interacting protein) is encoded by the PDCD6IP (also known as AIP1, ALIX, KIAA1375) gene (Gene ID 10015) in human. Alix, the mammalian orthologue of xenopus Xp95 and yeast BRO1, is an abundant adaptor protein involved in diverse cellular processes, including actin-based cytoskeleton assembly, integrin-mediated cell adhesion, and ESCRT- (endosomal sorting complex required for transport-) dependent membrane trafficking. Sorting of endocytosed membrane proteins by multivesicular bodies (MVBs) requires coordinated cargo recognition and assembly of membrane-sculpturing machinery that buds cargo-loaded membrane vesicles into the lumen of endosomes. MVB-mediated sorting is driven by five distinct ESCRT complexes (ESCRT-0, -I, -II and -III, and the Vps4 complex) and associated proteins. Alix and charged MVB protein 4 (CHMP4), the mammalian orthologue of yeast Snf7, are binding partners involved in a variety of ESCRT-III-mediated membrane-remodelling processes in mammalian cells, including retroviral budding, cytokinetic abscission, biogenesis of exosomes, plasma membrane repair, and ubiquitin-independent MVB sorting of protease-activated receptor 1 (PAR1). In the absence of binding partners, an intramolecular interaction between the Patch 2 region in the Bro1 domain (a.a. 3-392) and the TSG101-docking site (a.a. 717-720) in the Pro-rich domain (a.a. 717-860) locks Alix in a conformation inaccessible for interaction with CHMP4 and retroviral Gag proteins.

Clone 1A3 recognizes an epitope conserved among human Alix and xenopus Xp95, localized within the Bro1 domain C-terminal end Patch 2 region (Zhou, X., et al. (2009). Biochem. J. 418(2):277-284). Clone 1A3 immunostained WI-30 cells before, but not after, siRNA-mediated Alix knockdown (Pan, S., et al. (2008). EMBO J. 27(15):2077-2090). Epitope region is present in human Alix spliced isoform 1 and 2 (equivalent to mouse isoform 1 and 3, respectively), but absent in isoform 3 (equivalent to mouse isoform 2) reported by UniProt (Q8WUM4 for human and Q9WU78 for mouse).

Epitope: Patch 2 of the Bro1 domain.

GST-tagged recombinant full-length human Alix.

Anti-Alix Antibody, clone 1A3 is an antibody against Xp95 for use in Western Blotting, Immunoprecipitation, Immunocytochemistry.

Immunoprecipitation Analysis: A representative lot immunoprecipiated native Alix (1-709) GST fusion with C-terminal proline-rich domain (PRD; a.a. 710-868) deletion, but not native full-length Alix (1-868) GST fusion or native Alix (1-746) GST fusion with only partial PRD deletion (Zhou, X., et al. (2010). Biochem. J. 432(3):525-534).
Immunoprecipitation Analysis: A representative lot immunoprecipiated GST-Bro1 domain fragment, but not GST-full-length Alix protein under native conditions. Clone 1A3 immunoprecipitated full-length Alix from HEK293 lysates prepared with RIPA buffer, 1% NP40, or 0.1% SDS, but not from lysates prepared with TBS, 0.5% DOC, or 0.1% Triton X-100 (Zhou, X., et al. (2009). Biochem. J. 418(2):277-284).
Western Blotting Analysis: A representative lot detected wild-type human Alix and Xenopus Xp95 Bro1 domain GST fusions, but not the corresponding GST fusions with Y319F mutation in the Patch2 region (Zhou, X., et al. (2009). Biochem. J. 418(2):277-284).
Western Blotting Analysis: A representative lot detected recombinant xenopus Xp95 and human Alix GST fusion proteins, as well as endogenous Xp95 in extracts of both mature and immature Xenopus oocytes (Zhou, X., et al. (2009). Biochem. J. 418(2):277-284).
Western Blotting Analysis: A representative lot detected recombinant human Alix fragment a.a. 168-436, but not a.a. 436-709 (Pan, S., et al. (2008). EMBO J. 27(15):2077-2090).
Immunocytochemistry Analysis: A representative lot immunostained extracellular clumps and fibers by fluorescent immunocytochemistry staining of 4% paraformaldehyde-fixed, 0.5% Triton X-100-permeabilized WI-30 human diploid fetal lung fibroblasts. Alix immunostaining overlapped with that of anti-fibronectin, siRNA-mediated Alix knockdown eliminated the staining by clone 1A3 (Pan, S., et al. (2008). EMBO J. 27(15):2077-2090).
Note: In the native conformation of a full-length Alix protein, the Bro1 domain Patch 2 region is not exposed due to intramolecular interaction with the C-terminal proline-rich domain (PRD). RIPA buffer, 1% NP40, or 0.1% SDS (but not 0.5% DOC, or 0.1% Triton X-100) have been shownn to effectly alter Alix conformation and expose the Patch 2 region for antibody binding and protein interaction in immunoprecipitation and affinity interaction applications (Zhou, X., et al. (2009). Biochem. J. 418(2):277-284).

Research Category
Signaling

Research Sub Category
Vesicular Trafficking

Evaluated by Western Blotting in Daudi cell lysate.

Western Blotting Analysis: 0.5 µg/mL of a representative lot detected Alix in 10 µg of Daudi cell lysate.

~96 kDa observed. 96.02 kDa (human & mouse isoform 1), 96.77 kDa (human isoform 2 & mouse isoform 3) calculated. Uncharacterized band(s) may appear in some lysates.

Format: Purified

Protein G Purified

Purified mouse monoclonal IgG1κ antibody in buffer containing 0.1 M Tris-Glycine (pH 7.4), 150 mM NaCl with 0.05% sodium azide.

Stable for 1 year at 2-8°C from date of receipt.

Concentration: Please refer to lot specific datasheet.

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