Anti-Alix/Xp95, clone 1A3 (mouse monoclonal)

Species Reactivity	Key Applications	Host	Format	Antibody Type
H, Xn	WB, IP, ICC	M	Purified	Monoclonal Antibody

Species Reactivity

Key Applications

Host

Format

Antibody Type

H, Xn

WB, IP, ICC

Purified

Monoclonal Antibody

Description
Catalogue Number	MABS1277
Description	Anti-Alix/Xp95 Antibody, clone 1A3
Alternate Names	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
Background Information	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.

Description

Catalogue Number

MABS1277

Description

Anti-Alix/Xp95 Antibody, clone 1A3

Alternate Names

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

Background Information

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.

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

Product Information

Format

Purified

Presentation

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

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

Applications

Application

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

Key Applications

Western Blotting
Immunoprecipitation
Immunocytochemistry

Application Notes

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).

Biological Information
Immunogen	GST-tagged recombinant full-length human Alix.
Epitope	Patch 2 of the Bro1 domain.
Clone	1A3
Concentration	Please refer to lot specific datasheet.
Host	Mouse
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).
Isotype	IgG1κ
Species Reactivity	Human Xenopus
Species Reactivity Note	Human, Xenopus. Predicted to react with Mouse based on 100% sequence homology.
Antibody Type	Monoclonal Antibody
Entrez Gene Number	NP_037506
Gene Symbol	PDCD6IP AIP1 ALIX KIAA1375
Purification Method	Protein G Purified
UniProt Number	Q8WUM4
Molecular Weight	~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.

Biological Information

Immunogen

GST-tagged recombinant full-length human Alix.

Epitope

Patch 2 of the Bro1 domain.

Clone

1A3

Concentration

Please refer to lot specific datasheet.

Host

Mouse

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).

Isotype

IgG1κ

Species Reactivity

Human
Xenopus

Species Reactivity Note

Human, Xenopus. Predicted to react with Mouse based on 100% sequence homology.

Antibody Type

Monoclonal Antibody

Entrez Gene Number

NP_037506

Gene Symbol

PDCD6IP
AIP1
ALIX
KIAA1375

Purification Method

Protein G Purified

UniProt Number

Q8WUM4

Molecular Weight

~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.

Product Usage Statements
Quality Assurance	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.
Usage Statement	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.

Product Usage Statements

Quality Assurance

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.

Usage Statement

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.

Storage and Shipping Information
Storage Conditions	Stable for 1 year at 2-8°C from date of receipt.

Storage and Shipping Information

Storage Conditions

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

Packaging Information
Material Size	100 μg

Packaging Information

Material Size

100 μg

实验耗材

分子生物学试剂

蛋白与免疫学

生化试剂

细胞及检测试剂

色谱及层析

仪器设备

实验服务

办公用品

Anti-Alix/Xp95, clone 1A3 (mouse monoclonal)

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