MABS1277

Anti-Alix/Xp95 Antibody, clone 1A3

clone 1A3, from mouse

• Synonym(s): Programmed cell death 6-interacting protein, 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
• UNSPSC Code: 12352203
• eCl@ss: 32160702
• NACRES: NA.41

Properties

• biological source: mouse
• Quality Level: 100
• antibody form: purified immunoglobulin
• antibody product type: primary antibodies
• clone: 1A3, monoclonal
• species reactivity: human, Xenopus
• species reactivity (predicted by homology): mouse (based on 100% sequence homology)
• technique(s): immunocytochemistry: suitable; immunoprecipitation (IP): suitable; western blot: suitable
• isotype: IgG1κ
• NCBI accession no.: NP_037506
• UniProt accession no.: Q8WUM4
• target post-translational modification: unmodified
• Gene Information: human ... PDCD6IP(10015)

Description

General description

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.

Specificity

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).

Immunogen

Epitope: Patch 2 of the Bro1 domain.

GST-tagged recombinant full-length human Alix.

Application

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

Quality

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.

Target description

~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.

Physical form

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.

Storage and Stability

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

Other Notes

Concentration: Please refer to lot specific datasheet.

Disclaimer

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Safety Information

• Storage Class Code: 10 - Combustible liquids
• WGK: WGK 2
• Flash Point(F): Not applicable
• Flash Point(C): Not applicable