PDCD6IP Antibody
Product Specs
Target Background
ALIX (ALG-2-interacting protein X, also known as PDCD6IP) is a multifunctional protein with diverse roles in various cellular processes. It participates in endocytosis, multivesicular body (MVB) biogenesis, membrane repair, cytokinesis, apoptosis, and the maintenance of tight junction integrity. As a class E VPS protein, ALIX is involved in the concentration and sorting of cargo proteins within MVBs, facilitating their incorporation into intralumenal vesicles (ILVs). This process involves invagination and scission from the limiting membrane of the endosome. ALIX binds to lysobisphosphatidic acid (LBPA), a phospholipid abundant in MVB internal membranes. The MVB pathway requires the sequential action of ESCRT-O, -I, -II, and -III complexes. The ESCRT machinery also plays a crucial role in topologically similar membrane fission events, such as the final stages of cytokinesis. ALIX acts as an adapter protein for a subset of ESCRT-III proteins, such as CHMP4, mediating their function at specific membranes. It is essential for the completion of cytokinesis and may regulate both apoptosis and cell proliferation. In collaboration with syndecans (SDC1/4) and SDCBP, ALIX regulates exosome biogenesis. Through interactions with F-actin, PARD3, and TJP1, ALIX ensures the proper assembly and positioning of the actomyosin-tight junction complex at the apical sides of adjacent epithelial cells. This complex defines a crucial membrane domain for maintaining epithelial cell polarity and barrier function. In the context of microbial infection, ALIX is implicated in HIV-1 budding, potentially substituting for TSG101 in supporting viral release. This function necessitates interaction with CHMP4B. The ESCRT machinery is also involved in topologically equivalent membrane fission events, such as budding of enveloped viruses (including HIV-1 and other lentiviruses).
Selected Publications Highlighting ALIX Function:
- CD63-syntenin-1-ALIX complex regulation in post-endocytic HPV trafficking. PMID: 27578500
- ALIX and endophilin A in clathrin-independent endocytosis of cholera toxin and cell migration regulation. PMID: 27244115
- ALIX protein localization changes during colorectal adenoma-carcinoma progression. PMID: 27150162
- ALIX's role in glioma cell proliferation and its prognostic significance. PMID: 26980041
- ALIX in P2Y1 receptor degradation. PMID: 27301021
- K-Ras farnesylation and ALIX in extracellular nanovesicle packaging. PMID: 27909058
- ALIX's role in miRNA enrichment during extracellular vesicle biogenesis. PMID: 26935291
- HIV-1 nucleocapsid interaction with ALIX via syntenin. PMID: 26962944
- ARRDC3's role in ALIX ubiquitination induced by PAR1 activation. PMID: 26490116
- Phosphorylation of ALIX's PRD in ESCRT function activation. PMID: 26859355
- Bro1 domain binding specificity of Alix and Brox. PMID: 26866605
- ALIX depletion and abscission checkpoint signaling. PMID: 26929449
- PDCD6IP polymorphism and breast neoplasm risk. PMID: 26063962
- ALIX expression and Alzheimer's disease severity. PMID: 25502766
- Heparanase modulation of the syndecan-syntenin-ALIX pathway in exosome biogenesis. PMID: 25732677
- AIP1's role in suppressing tumor growth and metastasis. PMID: 26139244
- ALG-2, ALIX, and Vps4B in cell membrane shedding and repair. PMID: 25534348
- ALIX's role in multivesicular body sorting of membrane receptors. PMID: 25510652
- Aip1's role in actin filament severing and cytokinesis. PMID: 25451933
- ALIX in HIV-1 virion assembly and recycling. PMID: 24834918
- PDCD6IP polymorphism and non-small cell lung cancer susceptibility. PMID: 24870593
- HIV-1 Nef interaction with ALIX and CD4 lysosomal targeting. PMID: 25118280
- Syntenin-ALIX exosome biogenesis regulation by ARF6 and PLD2. PMID: 24637612
- ALIX's role in maintaining T84 monolayer barrier function. PMID: 24712823
- ALIX in cytokinesis, exosome release, and virus budding (Review). PMID: 24287454
- ALG-2's role as a calcium-dependent adapter bridging ALIX and ESCRT-I. PMID: 23924735
- PDCD6IP genetic variations and hepatocellular carcinoma risk. PMID: 23777424
- Alix as an adaptor for human parainfluenza virus type 1 interaction with the host ESCRT system. PMID: 23527201
- Lysobisphosphatidic acid recruitment of ALIX to late endosomes during viral infection. PMID: 23664863
- AP-3 facilitation of PAR1 interaction with ALIX. PMID: 22833563
- ALIX V domain binding to K63-linked polyubiquitin chains. PMID: 23201121
- BFRF1 recruitment of ESCRT components for Epstein Barr virus nuclear egress. PMID: 22969426
- BRCA2's influence on ESCRT-associated protein recruitment during abscission. PMID: 22771033
- Molecular recognition mechanisms between Src homology domain 3 and ALIX. PMID: 22641034
- Syndecan-syntenin-ALIX in membrane transport and signaling. PMID: 22660413
- Structural and functional analysis of the Bro1 domain in ALIX and HIV-1 budding. PMID: 22162750
- Alix Bro1 domain Phe105 loop mutations and HIV-1 release. PMID: 21889351
- ALIX V domain conformation and HIV-1 budding enhancement. PMID: 21715492
- Alix's role in hepatitis B virus capsid release. PMID: 21129143
- HIV-1 infection's effects on TSG101 and Alix expression. PMID: 21528537
- ALIX's role in Mopeia virus nucleoprotein incorporation into virus-like particles. PMID: 21248028
- Crystal structures revealing SIV Gag protein binding to ALIX. PMID: 20962096
- Intramolecular interactions regulating ALIX binding to ESCRT and viral proteins. PMID: 20929444
- ALIX colocalization with LBPA in DENV2-infected endothelial cells. PMID: 20669987
- Molecular recognition mechanisms between Src homology domain 3 and ALIX. PMID: 20670214
- ALG-2 isoform binding to Alix. PMID: 20691033
- Sendai virus M protein YLDL motif and Alix in virus budding. PMID: 20605035
- Alix recruitment of Nedd4-1 in HIV-1 release. PMID: 20519395
- p95's role in cell adhesion and morphology. PMID: 12360406
- AIP1/Alix interaction with ALG-2 and YPXL/I motif recognition. PMID: 12588984
Q&A
Factors Contributing to Discrepancies Between Observed and Calculated Molecular Weights in Western Blot
Observed molecular weights (MW) often differ from calculated values due to:
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Post-translational modifications (PTMs): Phosphorylation, ubiquitination, or glycosylation can increase apparent MW .
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Alternative splicing: PDCD6IP isoforms (e.g., truncated variants) may migrate at unexpected sizes .
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Protein-protein interactions: Stable complexes with ESCRT components (e.g., CHMP4B) may alter migration .
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Acrylamide gel composition: Higher acrylamide percentages improve resolution for smaller proteins but compress larger bands .
| Factor | Impact on MW | Mitigation Strategy |
|---|---|---|
| PTMs (e.g., phosphorylation) | ↑ Observed MW | Use phosphatase-treated lysates or PTM-specific antibodies |
| Alternative splicing | ↓ Observed MW | Include isoform-specific primers in qPCR controls |
| Protein complexes | ↑ Observed MW | Use reducing agents (e.g., β-mercaptoethanol) or heat denaturation |
For antibodies like ABclonal’s A2215 (observed 105 kDa vs. calculated 96 kDa), denaturing conditions (e.g., boiling lysates with SDS) are critical to disrupt interactions .
Optimizing Immunofluorescence Protocols for PDCD6IP Detection
PDCD6IP localizes to cytoplasmic structures (e.g., endosomes, centrosomes) . To enhance signal-to-noise ratio:
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Fixation: Use 4% PFA (freshly prepared) for membrane integrity; avoid methanol/acetone for cytoplasmic antigens .
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Permeabilization: 0.1–0.2% Triton X-100 for 10–15 minutes balances membrane access and structure preservation .
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Blocking: 3% BSA or 10% FBS in PBS-T (0.1% Tween) reduces nonspecific binding .
| Parameter | Recommended Setting | Rationale |
|---|---|---|
| Primary antibody | 1:100–1:200 (e.g., ABclonal A2215) | Balances sensitivity and background |
| Secondary antibody | DyLight®488-conjugated anti-rabbit | High photostability for confocal microscopy |
| Mounting medium | DAPI + vectashield® | Reduces photobleaching; nuclear counterstain |
For challenging samples (e.g., Jurkat cells), signal amplification (e.g., TSA kits) may improve detection .
Choosing Between Polyclonal and Monoclonal PDCD6IP Antibodies
| Type | Advantages | Disadvantages | Best Use Cases |
|---|---|---|---|
| Polyclonal (e.g., ABclonal A2215) | Broad epitope coverage; robust signal in diverse samples | Higher background risk | Initial screening, IHC, IF |
| Monoclonal (e.g., Sigma ZooMAb® 1D11) | High specificity; reproducible epitope targeting | Limited cross-reactivity | Quantitative WB, flow cytometry |
For apoptosis studies, polyclonal antibodies may capture multiple PDCD6IP isoforms, while monoclonals (e.g., targeting N-terminal regions) offer precision for ESCRT pathway analysis .
Addressing Cross-Reactivity in Predicted but Unconfirmed Species
When using antibodies validated for human/mouse/rat (e.g., Boster PB9770) on predicted species (e.g., pig), pre-adsorption with recombinant PDCD6IP or peptide blocking can reduce false positives . For flow cytometry, include isotype controls to distinguish true signal from nonspecific binding .
| Strategy | Procedure | Outcome |
|---|---|---|
| Peptide blocking | Incubate antibody with immunogen peptide (1:1 molar ratio) | Neutralizes specific epitope binding |
| Recombinant protein absorption | Pre-treat antibody with purified PDCD6IP (e.g., E. coli-derived) | Removes cross-reactive antibodies |
| Species-specific controls | Run parallel experiments with validated species lysates | Validates antibody performance |
Troubleshooting Unexpected Bands in Western Blot
Unexpected bands often arise from:
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Cross-reactivity: Antibodies binding to homologous proteins (e.g., ALG-2) .
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Secondary antibody nonspecificity: HRP conjugates binding to endogenous IgG .
For high-background membranes, reduce primary antibody incubation time or increase blocking duration .
Interpreting Antibody Performance in Single-Cell vs. Bulk Tissue Samples
Single-cell studies require antibodies with high signal sensitivity to detect low-abundance PDCD6IP. For example, Boster PB9770 (tested in Jurkat cells) may fail in single-cell RNA-seq workflows due to signal dilution . In contrast, Atlas Antibodies HPA051110 (validated via enhanced methods) may perform better under sparse conditions .
Advantages of Recombinant Monoclonal Antibodies
Recombinant monoclonals (e.g., Cusabio CSB-RA568053A0HU) offer:
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Consistent epitope targeting: Minimizes lot-to-lot variability .
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High affinity: Detects low-abundance PDCD6IP in flow cytometry .
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Reduced background: Engineered for minimal cross-reactivity .
For viral budding studies, recombinant antibodies may better resolve PDCD6IP interactions with ESCRT components .
Designing Experiments to Distinguish PDCD6IP’s Roles in Apoptosis vs. Endosomal Pathways
For calcium-dependent interactions, include EGTA (to chelate Ca²⁺) in lysate buffers .
Critical Factors in Choosing Secondary Antibodies
For multiplexing, use secondary antibodies with distinct fluorophores (e.g., Alexa Fluor® 488/647) .
