ARF1 Antibody

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Description

ARF1: Biological Role and Significance

ARF1 belongs to the ARF family of RAS superfamily GTPases, classified into three classes:

  • Class I: ARF1, ARF2, ARF3

  • Class II: ARF4, ARF5

  • Class III: ARF6

Key Functions:

  • Regulates vesicular trafficking and Golgi apparatus dynamics .

  • Activates phospholipase D and cholera toxin’s ADP-ribosyltransferase activity .

  • Modulates mitochondrial network connectivity, mitophagy, and oncogenic pathways (e.g., MAPK/ERK) .

ARF1 Antibody Applications

ARF1 antibodies are widely used in:

ApplicationDetails
Western Blot (WB)Detects ~18–21 kDa bands in human, mouse, rat, and monkey lysates .
Immunohistochemistry (IHC)Validated in breast, prostate, and colon cancer tissues .
Immunofluorescence (IF)Localizes ARF1 to Golgi apparatus and cytoplasmic vesicles .
Flow CytometryPermeabilization required for intracellular staining .
ELISAQuantifies ARF1 expression levels in cell lysates .

Cancer Biology

  • Breast Cancer:

    • ARF1 overexpression correlates with aggressive subtypes (HER2+, TNBC) and drives epithelial-mesenchymal transition (EMT) .

    • Knockdown reduces primary tumor growth and lung metastasis in murine models .

  • Prostate Cancer:

    • ARF1 activates the Raf1/MEK/ERK1/2 pathway, promoting tumorigenesis and anchorage-independent growth .

    • Silencing ARF1 inhibits cell proliferation and ERK1/2 activation in vivo .

  • Therapeutic Resistance:

    • ARF1 ablation induces mitochondrial stress, DAMPs release, and antitumor immune responses, sensitizing cancer stem cells to chemotherapy .

Cellular Trafficking

  • ARF1 compartments mature into recycling endosomes, directing post-Golgi cargo sorting .

  • AP-1 adaptor proteins regulate ARF1-mediated trafficking between Golgi and endosomes .

Validation and Specificity

  • Knockout Validation: ARF1 antibodies (e.g., ab58578) show no cross-reactivity in ARF1-knockout HeLa cells .

  • Subcellular Localization: Confocal microscopy confirms Golgi localization in prostate cancer cells .

  • Species Cross-Reactivity: Most antibodies recognize human, mouse, and rat ARF1, with limited reactivity in non-mammalian models .

Clinical and Therapeutic Implications

  • Biomarker Potential: High ARF1 expression in tumors predicts poor prognosis and correlates with T-cell exclusion .

  • Therapeutic Targeting: Inhibiting ARF1 disrupts oncogenic signaling (e.g., ERK1/2) and enhances chemotherapy efficacy .

Limitations and Considerations

  • Cross-Reactivity: Some antibodies may detect ARF2/ARF3 due to high sequence homology .

  • Sample Preparation: Optimal results require Triton X-100 permeabilization for intracellular staining .

Product Specs

Buffer
Preservative: 0.03% Proclin 300
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Form
Liquid
Lead Time
Made-to-order (14-16 weeks)
Synonyms
ARF1 antibody; ARF1A1C antibody; BEX1 antibody; At2g47170 antibody; T3D7.2 antibody; T8I13.1ADP-ribosylation factor 1 antibody; AtARF1 antibody; Protein BFA-VISUALIZED EXOCYTIC TRAFFICKING DEFECTIVE 1 antibody
Target Names
Uniprot No.

Target Background

Function
ARF1 is a GTP-binding protein crucial for protein trafficking. It plays a vital role in several cellular processes, including:
  • Sequence-specific vacuolar sorting to the lytic vacuole
  • ER-to-Golgi transport
  • Golgi-derived transport to the plasma membrane

Additionally, ARF1 is involved in:
  • Recruitment of COPI and GDAP1 to membranes
  • Recycling of PIN auxin transporters (e.g., PIN1 and PIN2) in a brefeldin A (BFA)-dependent manner
  • Various auxin-dependent developmental processes
Gene References Into Functions
  1. ARF1A1C is essential for recycling of PIN auxin transporters and for various auxin-dependent developmental processes. [BEX1][ARF1A1C] PMID: 24369434
Database Links

KEGG: ath:AT2G47170

STRING: 3702.AT2G47170.1

UniGene: At.12215

Protein Families
Small GTPase superfamily, Arf family
Subcellular Location
Golgi apparatus. Endosome. Golgi apparatus, trans-Golgi network. Early endosome.

Q&A

How do I select the most appropriate ARF1 antibody for Western blotting?

ARF1 antibodies vary in specificity, host species, and validated applications. Prioritize antibodies with:

  • Cross-reactivity confirmation: Ensure reactivity with the target species (e.g., human, mouse) via published data. For example, clone E01/8D1 (mouse monoclonal) detects a 19 kDa band in HEK293 lysates , while rabbit polyclonal antibody 20226-1-AP shows reactivity across DU145, HeLa, and HepG2 cells .

  • Application-specific validation: Antibodies validated for Western blotting (WB) may not perform optimally in immunohistochemistry (IHC) or immunoprecipitation (IP). The Proteintech antibody (20226-1-AP) supports WB, IP, IHC, and flow cytometry .

  • Batch consistency: Use vendors providing lot-specific validation data.

Table 1: ARF1 Antibody Performance Across Applications

Antibody CloneHostApplications (Validated)Observed MW (kDa)Key Citations
E01/8D1 (Bio-Rad)MouseWB, IP19Beck et al. 2008
20226-1-AP (Proteintech)RabbitWB, IP, IHC, IF/ICC, FC18–21Xu et al. 2017

What controls are essential for validating ARF1 antibody specificity?

  • Knockout/knockdown controls: Use ARF1-deficient cell lines (e.g., CRISPR-Cas9-generated AP1µA KO cells ) to confirm signal absence.

  • Blocking peptides: Pre-incubate antibodies with immunizing peptides to verify binding competition.

  • Orthogonal validation: Correlate antibody-derived data with ARF1 activation assays (e.g., GGA3 pull-downs or G-LISA® ).

How do I optimize ARF1 detection in immunohistochemistry?

  • Antigen retrieval: Use TE buffer (pH 9.0) or citrate buffer (pH 6.0) .

  • Titration: Start with 1:50–1:500 dilutions and adjust based on background noise .

  • Tissue-specific validation: Test antibody performance in ARF1-rich tissues (e.g., brain, liver) and pathological samples (e.g., breast cancer) .

How can I resolve discrepancies in ARF1 localization studies?

Conflicting reports of ARF1 localization (Golgi vs. cytoplasmic) often arise from:

  • Activation state: ARF1-GTP localizes to Golgi membranes, while ARF1-GDP is cytoplasmic . Use GTP-locked mutants or activation-specific assays (e.g., GGA3 pull-downs ) to contextualize findings.

  • Fixation artifacts: Methanol fixation preserves Golgi structures better than formaldehyde for IF/ICC .

  • Cell type variability: ARF1 compartments mature into recycling endosomes in some lineages (e.g., AP-1-dependent fission in HeLa cells ).

What methods quantify ARF1 activation in cancer models?

  • GTP-bound ARF1 pull-down assays: Incubate lysates with GGA3-PBD-conjugated beads to isolate active ARF1-GTP, followed by WB with ARF1 antibodies .

  • G-LISA®: A plate-based assay quantifying ARF1-GTP levels using absorbance (490 nm) . This method requires 1–5% of the lysate used in pull-down assays and is ideal for high-throughput screens .

  • Functional correlates: Link ARF1 activation to downstream pathways (e.g., mTORC1 in HNSCC) using γ-dipeptide inhibitors .

Table 2: Comparison of ARF1 Activation Assays

MethodSensitivitySample RequirementThroughputKey Advantage
GGA3 Pull-Down High500 µg proteinLowDirect GTP-binding validation
G-LISA® Moderate12.5 µg proteinHighQuantitative, suitable for 96-well format
IF/ICC with GTPase mutantsLowN/AMediumSpatial resolution of active ARF1

How do ARF1 inhibitors enhance chemotherapy efficacy?

  • Mechanism: γ-dipeptides (e.g., ATC-based compounds) block ARF1-GTP formation, disrupting Arf1-dependent trafficking and sensitizing cells to apoptosis .

  • Experimental design:

    • Treat HNSCC cells with inhibitors (72 hrs) in 2D/3D cultures.

    • Measure viability via ATP-based assays.

    • Validate ARF1 inactivation via GGA3 pull-downs and WB .

  • Data interpretation: Reduced ARF1 activation correlates with decreased PI3K/AKT signaling and increased caspase-3 cleavage .

How can I investigate ARF1’s role in neuronal migration disorders?

  • Model systems: Use iPSC-derived neurons from patients with ARF1 missense variants (e.g., Pro131Leu) .

  • Phenotypic assays:

    • Immunofluorescence: Assess periventricular nodular heterotopia (PVNH) using markers like DCX and TBR1 .

    • Live imaging: Track ARF1 compartment dynamics during neurite outgrowth .

  • Genotype-phenotype analysis: Switch-1 domain variants (e.g., Thr48Ala) may associate with milder cognitive deficits .

What strategies address contradictory ARF1 expression data across studies?

  • Meta-analysis controls: Normalize ARF1 levels to housekeeping genes (e.g., β-actin) using antibodies validated in parallel .

  • Contextual factors: Consider cell cycle stage, serum deprivation (activates ARF1 ), and tissue-specific isoforms.

  • Multiplex assays: Combine WB with RNA FISH to disentangle transcriptional vs. post-translational regulation.

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