ARF5 Antibody Pair

What is ARF5 and why is it important in research?

ARF5 belongs to the ARF family of small GTPases that regulate fundamental cellular functions. Recent research has shown that ARF5 is significantly correlated with poor prognosis in hepatocellular carcinoma (HCC) patients, suggesting it may function as an oncogene. ARF5 expression is significantly higher in HCC tissues compared to normal tissues, making it an important target for cancer research . Understanding ARF5's role in cellular processes has implications for developing targeted therapies and diagnostic tools in cancer research.

What species reactivity can be expected with ARF5 antibodies?

Commercial ARF5 antibodies, such as the polyclonal rabbit antibody (catalog # A05021), typically show reactivity to ARF5 in multiple species including human, mouse, and rat samples . This cross-reactivity is important for researchers conducting comparative studies across different model organisms. When selecting an antibody for your research, verify the specific reactivity profile to ensure compatibility with your experimental system.

What is the molecular weight of ARF5 protein in Western blot applications?

There is a notable discrepancy between the calculated and observed molecular weights of ARF5. While the calculated molecular weight is approximately 20.5 kDa, the observed molecular weight in Western blot applications is typically around 111 kDa . This difference could be due to post-translational modifications, protein complexes, or dimerization. When analyzing Western blot results, researchers should be aware of this discrepancy to correctly identify ARF5 bands.

What are the recommended protocols for ARF5 antibody use in Western blotting?

For Western blot applications, ARF5 antibodies are typically used at dilutions ranging from 1:1000 to 1:2000 . Optimal protocol includes:

• Sample preparation: Extract proteins using standard RIPA buffer with protease inhibitors

• Separation: Run 20-30 μg of protein on 10-12% SDS-PAGE

• Transfer: Use standard semi-dry or wet transfer to PVDF membrane

• Blocking: 5% non-fat milk in TBST for 1 hour at room temperature

• Primary antibody: Incubate with diluted ARF5 antibody (1:1000-1:2000) overnight at 4°C

• Secondary antibody: Anti-rabbit HRP-conjugated antibody (1:5000-1:10000)

• Detection: Use ECL substrate and imaging system

Optimization may be required based on specific sample types and experimental conditions.

How should ARF5 antibodies be stored and handled to maintain reactivity?

For optimal performance, ARF5 antibodies should be stored at -20°C for long-term storage (up to one year). For frequent use and short-term storage (up to one month), 4°C is recommended . Avoid repeated freeze-thaw cycles as they can compromise antibody functionality. ARF5 antibodies are typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.2 , which helps maintain stability. Always centrifuge briefly before opening the vial to ensure all liquid is at the bottom.

What validation methods are recommended to confirm ARF5 antibody specificity?

Comprehensive validation of ARF5 antibodies should include:

• Positive and negative control samples (tissues or cell lines with known expression levels)

• Knockdown/knockout validation using siRNA or CRISPR techniques

• Peptide competition assays using the immunizing peptide

• Cross-validation with multiple antibodies targeting different epitopes

• Testing across multiple applications (WB, IHC, ICC, IF) if claimed by manufacturer

Reputable antibody manufacturers validate their products through Western blot, immunohistochemistry, immunocytochemistry, immunofluorescence, and ELISA with both positive control and negative samples to ensure specificity and high affinity .

What is the relationship between ARF5 and immune cell infiltration in tumors?

ARF5 shows significant correlation with immune cell infiltration in hepatocellular carcinoma. Analysis indicates a strong positive correlation between ARF5 expression and CD4+ T cells (R = 0.4, p < 2.2e-16), and weaker correlations with invasive memory B cells (R = 0.2, p = 5.4e-05), neutrophils (R = 0.13, p = 0.0078), macrophages (R = 0.11, p = 0.028), and myeloid dendritic cells (R = 0.28, p = 4.6e-9) . Interestingly, ARF5 shows a negative correlation with CD8+ T cells (R = -0.11, p = 0.022). These findings suggest ARF5 might promote immune escape of tumor cells by influencing CD4+ T cell activation and other immune components .

What pathways are associated with ARF5 expression in hepatocellular carcinoma?

Gene Set Enrichment Analysis (GSEA) reveals that ARF5 expression in HCC positively correlates with pathways involved in:

• RNA synthesis and translation processes

• Splice complexes formation

• Spliceosome assembly

• Regulation of actin cytoskeleton

Pathways negatively correlated with ARF5 expression include:

• Amino acid metabolism

• Lipid metabolism

These pathway associations suggest ARF5 may influence cancer progression through effects on RNA processing and cytoskeletal regulation, while potentially altering cellular metabolism .

How can WGCNA be utilized to identify ARF5-associated gene networks?

Weighted Gene Co-expression Network Analysis (WGCNA) can be employed to identify ARF5-related gene networks in cancer research using the following methodology:

• Data preprocessing: Select 25% of the variance of differentially expressed genes (DEGs)

• Similarity matrix construction: Calculate Pearson correlation coefficients between gene pairs

• Adjacency matrix transformation: Convert similarity matrix to adjacency matrix

• Topological overlap matrix (TOM) creation: Transform adjacency matrix to describe association strength between genes

• Module identification: Perform hierarchical cluster analysis using TOM as input

• Module-trait correlation: Analyze correlation between identified modules and tumor/normal status

• Module-ARF5 correlation: Identify modules with highest correlation to ARF5 expression

In HCC research, the blue module showed significant correlation with both HCC status and ARF5 expression, making it valuable for further biological function network analysis .

What approaches can be used to investigate miRNA regulation of ARF5 expression?

Investigation of miRNA regulation of ARF5 requires a multi-faceted approach:

• Bioinformatic prediction: Use databases like TargetScan, miRDB, and miRWalk to identify miRNAs with potential binding sites in ARF5 mRNA

• Expression correlation analysis: Analyze negative correlation between miRNA candidates and ARF5 expression in tissue samples

• Luciferase reporter assays: Clone ARF5 3'UTR into reporter vectors to validate direct binding

• Gain/loss-of-function studies: Overexpress or inhibit candidate miRNAs to assess effects on ARF5 expression

• ceRNA network analysis: Identify competing endogenous RNAs (lncRNAs, circRNAs) that may sponge miRNAs targeting ARF5

Research has identified miR-29 as a potential regulator of ARF5 expression in HCC, with 138 ceRNAs potentially promoting ARF5 expression through competitive binding of miR-29 .

How can ARF5's role in the tumor immune microenvironment be assessed?

To comprehensively evaluate ARF5's role in the tumor immune microenvironment (TIME), researchers should implement:

• Cell type-specific expression analysis: Utilize databases like Human Protein Atlas to assess ARF5 expression across liver cells and immune cells

• Immune infiltration correlation: Analyze associations between ARF5 expression and immune cell infiltration using databases like ImmuCellAI and TIMER2

• Single-cell RNA sequencing: Perform scRNA-seq to identify cell-specific expression patterns and intercellular communication

• Functional validation: Use co-culture systems with immune and cancer cells, manipulating ARF5 expression

• Cytokine/chemokine profiling: Measure changes in immune signaling molecules upon ARF5 modulation

• In vivo immune depletion studies: Assess ARF5's effects in immunodeficient or immune cell-depleted animal models

Research indicates ARF5 expression is higher in immune cells (T cells and B cells) than in liver cells, suggesting immunological relevance that may impact immunotherapy responsiveness in HCC patients .

How to address the discrepancy between calculated and observed molecular weights for ARF5?

The significant difference between ARF5's calculated molecular weight (20.5 kDa) and observed molecular weight (111 kDa) requires careful troubleshooting:

• Verify antibody specificity using positive and negative controls

• Assess post-translational modifications (phosphorylation, glycosylation, ubiquitination) that may alter migration

• Evaluate protein-protein interactions or complex formation that persist despite denaturing conditions

• Test different sample preparation methods (varying detergents, reducing agents)

• Run gradient gels to improve resolution

• Consider using mass spectrometry to confirm protein identity

If the discrepancy persists across multiple experimental conditions and verification methods, it may represent a biological reality for ARF5 in certain contexts that warrants further investigation.

What considerations are important when analyzing ARF5's impact on immune cell infiltration?

When analyzing ARF5's associations with immune infiltration, researchers should consider:

• Tissue heterogeneity: Account for variations in cellular composition across tumor samples

• Correlation vs. causation: Establish whether ARF5 directly influences immune cell recruitment or if correlations are secondary to other factors

• Cell type specificity: Differentiate between immune cell subtypes (e.g., CD4+ T cell subpopulations)

• Spatial distribution: Assess whether immune cells are located intratumorally or in the periphery

• Functional states: Determine activation/exhaustion status of immune cells

• Confounding factors: Control for tumor stage, grade, treatment history, and patient demographics

Research indicates varying strength of correlations between ARF5 and different immune cell types, with strongest positive correlation to CD4+ T cells (R = 0.4) and negative correlation to CD8+ T cells (R = -0.11) , suggesting complex immunoregulatory mechanisms.

How can researchers integrate ARF5 findings with current immunotherapy approaches?

Integration of ARF5 research with immunotherapy approaches requires:

• Stratification analysis: Evaluate if ARF5 expression levels predict response to immunotherapy

• Combination strategies: Test whether ARF5 inhibition enhances efficacy of immune checkpoint inhibitors

• Biomarker development: Assess ARF5 expression in liquid biopsies as a non-invasive biomarker

• Pathway targeting: Develop approaches targeting ARF5-regulated pathways that influence immune responses

• Single-cell analysis: Perform scRNA-seq on pre- and post-treatment samples to track ARF5's impact on immune landscape

Given that individual differences between patients limit immunotherapy benefits, and ARF5 shows significant correlation with immune cell infiltration in HCC, this protein may represent an important factor in understanding and improving immunotherapy responsiveness .