ARL6IP6 Antibody

What is ARL6IP6 and what is its relevance in research?

ARL6IP6 (ADP-ribosylation factor-like protein 6-interacting protein 6) is a member of the ADP ribosylation factor (like) protein family that has attracted significant research interest in recent years. It functions as a potential therapeutic target and prognostic biomarker in various malignancies, with particular significance in hepatocellular carcinoma (HCC) . ARL6IP6 has been shown to be differentially expressed across multiple cancer types, with notably elevated expression in liver cancer tissue compared to healthy liver tissue . The protein has a molecular weight of approximately 22 kDa and has been detected in various human tissues and cell lines . Understanding ARL6IP6 expression patterns and functions may provide valuable insights into cancer pathogenesis and potential therapeutic interventions.

What detection methods are available for ARL6IP6 research?

ARL6IP6 can be detected using multiple methodological approaches, each with specific advantages depending on your research questions:

• Immunohistochemistry (IHC): Effective for visualizing ARL6IP6 expression in paraffin-embedded tissue sections. Validated protocols typically use heat-mediated antigen retrieval in EDTA buffer (pH 8.0) with antibody concentrations of 1-2 μg/ml or dilutions of 1:200-1:500 .

• Western Blotting: Detects ARL6IP6 protein at approximately 22 kDa, with recommended antibody concentrations of 0.04-0.4 μg/mL or 0.5 μg/mL depending on the specific antibody .

• Immunofluorescence (IF): Enables subcellular localization studies with recommended antibody concentrations of 0.25-2 μg/mL or 5 μg/mL depending on the antibody source .

• Flow Cytometry: Allows quantitative analysis of ARL6IP6 expression at the single-cell level, typically using 1 μg antibody per million cells after appropriate fixation and permeabilization .

• ELISA: Quantitative detection in solution with sensitivity down to 1.0 pg/mL and detection range of 50-1000 pg/mL for commercially available kits .

What sample types are suitable for ARL6IP6 detection?

ARL6IP6 can be successfully detected in multiple sample types, allowing for diverse experimental approaches:

• Cell lines: Validated in human 293T, Caco-2, and HEPA1-6 cells .

• Tissue samples: Successfully detected in human cancer tissues (bladder, gastric, lung, and ovarian), as well as rodent tissues including rat lung and mouse testis and lung .

• Liquid biopsies: Detection possible in cell culture supernatant, plasma, serum, and tissue homogenates using ELISA methodology .

• Paraffin-embedded sections: Effective for immunohistochemical analysis with appropriate antigen retrieval techniques .

For optimal results with any sample type, fresh samples without long-term storage are recommended to prevent protein degradation and denaturation .

What are the recommended experimental conditions for ARL6IP6 antibody applications?

For Western blotting:

• Protein loading: 30 μg of sample under reducing conditions

• Antibody concentration: 0.5 μg/mL incubated overnight at 4°C

• Secondary antibody: Anti-rabbit IgG-HRP at 1:5000 dilution

• Signal development: Enhanced chemiluminescent detection system

For immunohistochemistry:

• Antigen retrieval: Heat-mediated in EDTA buffer (pH 8.0)

• Blocking: 10% goat serum

• Primary antibody: 2 μg/ml incubated overnight at 4°C

• Secondary detection: Biotinylated goat anti-rabbit IgG with Streptavidin-Biotin-Complex and DAB chromogen

For immunofluorescence:

• Cell preparation: Enzyme antigen retrieval

• Blocking: 10% goat serum

• Primary antibody: 5 μg/mL incubated overnight at 4°C

• Secondary detection: Fluorescent-conjugated anti-rabbit IgG at 1:100 dilution

• Counterstaining: DAPI for nuclear visualization

How does ARL6IP6 expression correlate with cancer progression and patient outcomes?

Research has revealed significant correlations between ARL6IP6 expression and cancer progression, particularly in hepatocellular carcinoma:

These findings suggest ARL6IP6 may serve as a valuable prognostic biomarker in HCC and potentially other malignancies, with higher expression levels generally associated with more aggressive disease and poorer outcomes.

What is the relationship between ARL6IP6 and tumor-infiltrating immune cells?

Recent research has demonstrated a significant relationship between ARL6IP6 expression and the tumor immune microenvironment:

• Positive correlations: ARL6IP6 expression levels positively correlate with the activities of multiple tumor-infiltrating immune cells, including:

  • B cells

  • Myeloid dendritic cells

  • Macrophages

  • Neutrophils

  • CD8+ T cells

  • CD4+ T cells

This association suggests ARL6IP6 may play a role in modulating the tumor immune microenvironment, potentially influencing immune surveillance and response to immunotherapy. Researchers investigating cancer immunology should consider ARL6IP6 as a potential factor influencing immune cell recruitment or activity within the tumor microenvironment.

How can I validate ARL6IP6 antibody specificity in my experimental system?

Validating antibody specificity is crucial for obtaining reliable research results. For ARL6IP6 antibodies, consider these validation approaches:

• Protein knockdown/knockout controls: Compare staining between wild-type samples and those with ARL6IP6 knockdown/knockout to confirm signal specificity.

• Multiple antibody validation: Use antibodies from different sources or those targeting different epitopes of ARL6IP6 to confirm consistent staining patterns.

• Enhanced validation methods: Look for antibodies validated through orthogonal RNAseq or recombinant expression techniques, which provide higher confidence in specificity .

• Multi-tissue Western blotting: Analyze expression across multiple tissues with known differential expression patterns of ARL6IP6, such as comparing liver cancer tissue (high expression) with other cancer types showing lower expression .

• Immunogen competition assay: Pre-incubate the antibody with its immunogen peptide (such as the sequence MSFAESGWRSALRRRGPGTPGPVARPSYSSFTQGDSWGEGEVDEEEGCDQVARDLRAEFSAGAWSEPRKRSVLPPDGNGSPVLPDKRNGIFP) to demonstrate signal reduction .

What are the optimal protocols for multiplexing ARL6IP6 with other markers?

When designing multiplexed staining protocols including ARL6IP6, consider these methodological approaches:

• Sequential immunofluorescence:

  • Begin with the most sensitive antigen (typically ARL6IP6)

  • Use primary antibodies from different host species to avoid cross-reactivity

  • Employ spectrally distinct fluorophores for each marker

  • Include appropriate blocking steps between antibody applications

  • Consider tyramide signal amplification for weaker signals

• Panel design considerations:

  • When studying immune cell interactions, combine ARL6IP6 with markers for B cells, myeloid dendritic cells, macrophages, neutrophils, CD8+T cells, or CD4+T cells based on the positive correlations observed in research

  • For cancer progression studies, consider combining with TP53 markers given the relationship between ARL6IP6 expression and TP53 mutation status

• Chromogenic multiplexing:

  • Use different chromogens (DAB, red chromogen, etc.)

  • Perform sequential staining with complete stripping or blocking between rounds

  • Include single-stained controls for each marker to validate specificity

What approaches should be used for troubleshooting inconsistent ARL6IP6 staining?

When encountering variable or inconsistent staining results with ARL6IP6 antibodies, consider these troubleshooting approaches:

• Tissue-specific optimization:

  • Different cancer tissues may require specific antigen retrieval conditions

  • For challenging tissues, test both EDTA (pH 8.0) and citrate buffer (pH 6.0)

  • Adjust antibody concentration based on tissue type (1:200-1:500 dilution range)

• Fixation considerations:

  • Overfixation may mask epitopes; optimize fixation time

  • For cell lines, compare 4% paraformaldehyde fixation with methanol fixation

  • Fresh samples yield better results than long-stored samples

• Signal amplification strategies:

  • For weak signals, implement tyramide signal amplification

  • Use polymer-based detection systems instead of standard secondary antibodies

  • Consider biotin-streptavidin systems as demonstrated in successful IHC protocols

• Blocking optimization:

  • Increase blocking concentration (test 10-15% serum)

  • Include protein blockers like BSA or casein

  • Add detergents to reduce background (0.1-0.3% Triton X-100 or Tween-20)

• Control inclusion:

  • Always run known positive controls (such as liver cancer tissue)

  • Include technical negative controls (omitting primary antibody)

  • Consider biological negative controls (tissues with minimal ARL6IP6 expression)

What are the critical factors for successful Western blot detection of ARL6IP6?

Western blotting for ARL6IP6 requires attention to several critical factors to ensure reliable detection of this approximately 22 kDa protein:

• Sample preparation optimization:

  • Use RIPA buffer with protease inhibitors for efficient extraction

  • Load adequate protein (30 μg recommended) under reducing conditions

  • Run on 5-20% SDS-PAGE gels at 70V (stacking)/90V (resolving) for optimal separation

• Transfer parameters:

  • Transfer to nitrocellulose membrane at 150 mA for 50-90 minutes

  • Verify transfer efficiency with reversible protein stains before blocking

• Antibody conditions:

  • Block with 5% non-fat milk/TBS for 1.5 hours at room temperature

  • Incubate with primary antibody at 0.5 μg/mL overnight at 4°C

  • Wash thoroughly with TBS-0.1% Tween (3 times, 5 minutes each)

  • Use goat anti-rabbit IgG-HRP secondary at 1:5000 dilution for 1.5 hours at room temperature

• Expected results interpretation:

  • Primary band should appear at approximately 22 kDa

  • Expression varies by tissue type, with detectable signals in human 293T and Caco-2 cells, rat lung tissue, and mouse testis and lung tissue

How can I optimize immunohistochemical detection of ARL6IP6 in different cancer tissues?

Successful immunohistochemical detection of ARL6IP6 across diverse cancer tissues requires thoughtful optimization:

• Tissue-specific antigen retrieval:

  • Heat-mediated antigen retrieval in EDTA buffer (pH 8.0) has shown success across multiple cancer tissues

  • Extend retrieval time for heavily fixed tissues (15-20 minutes)

• Blocking and antibody parameters:

  • Block sections with 10% goat serum to minimize background

  • Apply primary antibody at 2 μg/ml concentration

  • Incubate overnight at 4°C for optimal sensitivity

  • Use biotinylated goat anti-rabbit IgG as secondary antibody (30 minutes at 37°C)

  • Develop with Strepavidin-Biotin-Complex and DAB chromogen

• Cancer-specific considerations:

  • Validated protocols exist for bladder epithelial carcinoma, gastric cancer, lung cancer, ovarian cancer, and metaplasia of squamous cells of the renal pelvis

  • Expression patterns vary by cancer type, with particularly strong expression in hepatocellular carcinoma

  • Consider double-staining with cancer-specific markers when analyzing novel tissue types

• Controls and interpretation:

  • Include paired normal tissue controls when possible

  • Compare staining patterns with established expression data from databases like GEPIA and UALCAN

  • Correlate with cancer stage and grade information for prognostic interpretation

What are the best approaches for quantitative analysis of ARL6IP6 expression data?

For rigorous quantitative analysis of ARL6IP6 expression data across different experimental platforms:

• IHC quantification methods:

  • Use digital pathology software for unbiased scoring

  • Implement H-score system (intensity × percentage of positive cells)

  • Alternatively, use Allred score combining intensity and proportion

  • Set thresholds based on correlation with clinical outcomes

• Western blot quantification:

  • Normalize ARL6IP6 signal to loading controls (β-actin, GAPDH)

  • Use densitometry software with local background subtraction

  • Generate standard curves with recombinant protein for absolute quantification

• ELISA-based quantification:

  • Utilize the quantitative range of 50-1000 pg/mL

  • Include all standards (A-F) for accurate calibration curves

  • Calculate concentrations using four-parameter logistic regression

• Transcriptomic data analysis:

  • Compare expression across cancer databases (TCGA, GEPIA)

  • Correlate with survival data using Kaplan-Meier analysis

  • Implement Cox regression for multivariate analysis with other prognostic factors

How can I effectively use ARL6IP6 antibodies in flow cytometry applications?

Optimizing flow cytometry protocols for ARL6IP6 detection requires specific technical considerations:

• Cell preparation protocol:

  • Fix cells with 4% paraformaldehyde

  • Permeabilize with dedicated permeabilization buffer

  • Block with 10% normal goat serum to reduce non-specific binding

• Antibody titration and application:

  • Use 1 μg antibody per 1×10^6 cells as a starting concentration

  • Optimize through titration experiments

  • Include isotype controls at matching concentrations

• Gating strategy:

  • Use forward and side scatter to identify cell populations

  • Apply appropriate fluorescence-minus-one (FMO) controls

  • Create overlay histograms to visualize ARL6IP6 expression distribution

• Multiparameter analysis:

  • Consider co-staining with markers of interest based on research into ARL6IP6's relationship with immune cells

  • Apply compensation controls when using multiple fluorophores

  • Analyze correlations between ARL6IP6 expression and other cellular parameters

What is the potential of ARL6IP6 as a therapeutic target in cancer research?

ARL6IP6 shows promising characteristics as a potential therapeutic target based on current research findings:

Future research should focus on mechanistic studies to better understand how ARL6IP6 contributes to cancer pathogenesis and validate its potential as a therapeutic target through preclinical models.

What methods should be used to investigate the functional role of ARL6IP6 in cancer cell biology?

To elucidate the functional significance of ARL6IP6 in cancer biology, researchers should consider these methodological approaches:

• Gene modulation techniques:

  • CRISPR/Cas9 knockout to assess loss-of-function effects

  • shRNA or siRNA for transient knockdown studies

  • Overexpression systems to evaluate gain-of-function effects

  • Inducible expression systems for temporal control

• Functional assays:

  • Cell proliferation assessment using CCK8 assay

  • Apoptosis quantification via flow cytometry

  • Cell migration and invasion evaluation with transwell assays

  • Cell cycle analysis to determine effects on cell division

• Pathway analysis:

  • Protein interaction studies (co-IP, proximity labeling)

  • Phosphorylation state analysis of downstream effectors

  • Transcriptomic profiling after ARL6IP6 modulation

  • Investigation of connections to TP53 pathway given correlation with TP53 mutation status

• In vivo models:

  • Xenograft studies with ARL6IP6-modulated cell lines

  • Patient-derived xenografts to preserve tumor heterogeneity

  • Analysis of immune cell infiltration in immunocompetent models

  • Correlation of treatment response with ARL6IP6 expression levels