ARL6IP4 Antibody

What is ARL6IP4 and why is it studied?

ARL6IP4 (ADP-Ribosylation-Like Factor 6 Interacting Protein 4) functions as a splicing regulator within nuclear speckles and nucleoli. Research indicates it interacts with SC35 and modulates alternative pre-mRNA splicing in vivo . The protein contains multiple serine-rich regions and has been implicated in RNA processing pathways. Understanding its function is valuable for investigating gene expression regulation mechanisms, particularly in the context of splicing regulation and nuclear organization. Studies utilizing ARL6IP4 antibodies have contributed to elucidating its subcellular localization and protein-protein interactions.

What types of ARL6IP4 antibodies are available for research applications?

ARL6IP4 antibodies are available in several configurations based on host organism, clonality, and epitope recognition. Common variants include:

• Mouse polyclonal antibodies raised against full-length human ARL6IP4 (AA 1-360)

• Rabbit polyclonal antibodies targeting specific regions such as the middle region

• Mouse monoclonal antibodies (e.g., clone 5E5) against specific epitopes

• Species-specific antibodies with varied cross-reactivity profiles against human, monkey, cow, horse, pig, dog, and rat ARL6IP4

The choice of antibody depends on experimental requirements and target species, with options for unconjugated formats optimized for various applications including Western blotting, immunofluorescence, ELISA, and immunohistochemistry.

What are the primary applications for ARL6IP4 antibodies?

ARL6IP4 antibodies are validated for multiple experimental applications:

• Western Blotting (WB): For detecting denatured ARL6IP4 protein in cell and tissue lysates, allowing quantification and molecular weight confirmation

• Immunofluorescence (IF): For visualizing subcellular localization of ARL6IP4, particularly its distribution in nuclear speckles and nucleoli

• Immunoprecipitation (IP): For isolating ARL6IP4 protein complexes to study protein-protein interactions

• ELISA: For quantitative measurement of ARL6IP4 protein levels

• Immunohistochemistry (IHC): For detecting ARL6IP4 expression in tissue sections, enabling analysis of expression patterns across different cell types

Selection of the appropriate application should align with research objectives and be supported by validated antibody performance in the specific experimental context.

How should researchers select the appropriate ARL6IP4 antibody for their experiments?

When selecting an ARL6IP4 antibody, researchers should consider:

• Experimental application (WB, IF, IHC, IP, or ELISA)

• Target species and cross-reactivity requirements

• Epitope recognition and binding specificity

• Host species compatibility with secondary detection systems

• Clonality (monoclonal for specificity or polyclonal for sensitivity)

• Validation data supporting performance in the intended application

For example, studies focused on human samples would benefit from antibodies with demonstrated human reactivity, while cross-species studies might require antibodies with broader species recognition profiles such as ABIN2790727, which reacts with human, cow, horse, pig, dog, and rat ARL6IP4 . Additionally, the specific region of interest within the protein should guide epitope selection, with options ranging from full-length coverage (AA 1-360) to specific domains.

How do different epitope-targeting strategies affect ARL6IP4 antibody performance in detecting protein isoforms?

ARL6IP4 antibodies targeting different epitopes exhibit varying capacities to detect specific protein isoforms and post-translationally modified forms. Antibodies recognizing the full-length protein (AA 1-360) provide comprehensive detection but may not distinguish between isoforms . Conversely, region-specific antibodies enable more targeted analysis:

• N-terminal antibodies (AA 1-50): Effective for detecting most isoforms but may miss N-terminally truncated variants

• Middle region antibodies (e.g., ABIN2790727): Particularly useful for detecting the conserved functional domains containing serine-rich regions important for splicing regulation

• C-terminal antibodies (AA 310-360): Valuable for isoforms with conserved C-termini

For studies investigating alternative splicing of ARL6IP4 itself, employing multiple antibodies targeting different protein regions is recommended to comprehensively characterize expression patterns and potential functional variations. This approach has revealed that certain isoforms may localize differently within nuclear subcompartments, suggesting distinct functional roles.

What are the optimal experimental conditions for detecting ARL6IP4 interactions with other splicing factors?

Detecting ARL6IP4 interactions with splicing factors such as SC35 requires careful experimental design:

• Buffer composition is critical - use buffers containing 20mM HEPES (pH 7.9), 150mM KCl, 0.2mM EDTA, 10% glycerol, and 1mM DTT supplemented with phosphatase and protease inhibitors to preserve interactions

• For co-immunoprecipitation experiments:

  • Pre-clear lysates with appropriate control IgG

  • Use antibodies against full-length ARL6IP4 (AA 1-360) for broader interaction detection

  • Consider mild crosslinking (0.1-0.5% formaldehyde) to stabilize transient interactions

  • Include RNase treatment controls to distinguish RNA-dependent from direct protein-protein interactions

• For proximity ligation assays:

  • Optimize fixation conditions (4% paraformaldehyde for 10-15 minutes)

  • Use antibodies raised in different host species against ARL6IP4 and potential partners

  • Include appropriate negative controls with single antibodies

Published research indicates that interactions between ARL6IP4 and other splicing regulators occur primarily in nuclear speckles, requiring careful subcellular fractionation and imaging approaches for accurate characterization.

How can researchers distinguish between the different functional domains of ARL6IP4 using domain-specific antibodies?

ARL6IP4 contains distinct functional domains that can be investigated using domain-specific antibodies:

• The N-terminal region (AA 1-50): Contains regulatory elements for nuclear localization

• The serine-rich domain (middle region): Critical for interactions with other splicing factors

• The C-terminal region (AA 310-360): Involved in protein stability and additional protein-protein interactions

For domain-specific functional studies:

• Use immunofluorescence with domain-specific antibodies to map localization patterns

• Combine with deletion mutant expression to validate domain functions

• Employ domain-specific antibodies in ChIP-seq or CLIP-seq experiments to identify domain-specific RNA or DNA interactions

• Use competition assays with purified protein domains to validate epitope specificity

This approach has revealed that the serine-rich middle region of ARL6IP4 is particularly important for its role in splicing regulation, as antibodies targeting this region can disrupt interactions with other splicing factors in reconstituted splicing assays.

What are the critical parameters for optimizing Western blot detection of ARL6IP4?

Successful Western blot detection of ARL6IP4 requires careful optimization of multiple parameters:

• Sample preparation:

  • Use RIPA buffer (50mM Tris-HCl pH 7.4, 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitors

  • Include phosphatase inhibitors to preserve post-translational modifications

  • Heat samples at 95°C for 5 minutes in reducing sample buffer

• Gel electrophoresis and transfer:

  • Use 10-12% polyacrylamide gels for optimal resolution

  • Transfer to PVDF membranes at 100V for 60-90 minutes in cold transfer buffer

  • Verify transfer efficiency with reversible protein stain

• Antibody incubation:

  • Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

  • For antibody ABIN949309, use 1:1000-1:2000 dilution in 5% BSA/TBST overnight at 4°C

  • For antibody ABIN2790727, use 1:500-1:1000 dilution in 5% BSA/TBST overnight at 4°C

  • Wash extensively with TBST (4-5 times, 5 minutes each)

• Detection specifics:

  • ARL6IP4 typically appears as a band of approximately 42-45 kDa

  • Additional bands may represent isoforms or post-translationally modified forms

  • Include positive controls (e.g., cell lines with known ARL6IP4 expression)

This methodological approach ensures consistent and specific detection of ARL6IP4 in various sample types, minimizing background and enhancing reproducibility.

How should researchers optimize immunofluorescence protocols for ARL6IP4 subcellular localization studies?

For successful immunofluorescence detection of ARL6IP4's nuclear speckle localization:

• Sample preparation:

  • Grow cells on appropriate coverslips to 70-80% confluence

  • Fix with 4% paraformaldehyde for 15 minutes at room temperature

  • For nuclear antigen accessibility, permeabilize with 0.2% Triton X-100 for 10 minutes

• Antibody incubation:

  • Block with 3% BSA in PBS for 1 hour at room temperature

  • Incubate with primary antibody (1:100-1:500 dilution) in blocking buffer overnight at 4°C

  • Wash 3-4 times with PBS (5 minutes each)

  • Incubate with appropriate fluorophore-conjugated secondary antibody (1:500-1:1000) for 1 hour at room temperature

  • Include DAPI (1:1000) for nuclear counterstaining

• Co-localization studies:

  • For nuclear speckle visualization, co-stain with antibodies against SC35 or other splicing factors

  • Use different host species antibodies to allow simultaneous detection

  • Analyze co-localization using appropriate quantification tools (e.g., Pearson's correlation coefficient)

• Controls and validation:

  • Include secondary-only controls to assess background

  • Use siRNA-mediated knockdown of ARL6IP4 as negative control

  • Consider peptide competition assays to verify specificity

This approach enables precise characterization of ARL6IP4's subnuclear distribution and its co-localization with other splicing regulatory factors.

What considerations are important when designing immunoprecipitation experiments with ARL6IP4 antibodies?

Effective immunoprecipitation of ARL6IP4 and its interaction partners requires:

• Lysis buffer optimization:

  • Use gentle, non-denaturing lysis buffer (20mM Tris-HCl pH 7.5, 150mM NaCl, 1mM EDTA, 1% NP-40, 5% glycerol) with protease/phosphatase inhibitors

  • For nuclear protein extraction, include nuclear extraction steps with appropriate buffers

• Antibody selection and protocol:

  • Pre-clear lysates with Protein A/G beads to reduce non-specific binding

  • Use 2-5 μg of antibody per 500 μg of total protein

  • Incubate with antibody overnight at 4°C with gentle rotation

  • Add pre-washed Protein A/G beads and incubate for 1-3 hours at 4°C

  • Wash extensively (4-5 times) with cold lysis buffer

• Elution and analysis:

  • Elute with 2X SDS sample buffer at 95°C for 5 minutes

  • Analyze by Western blot, mass spectrometry, or other downstream applications

  • Include IgG control from the same species as the primary antibody

• Validation strategies:

  • Confirm successful IP by Western blotting a small fraction for ARL6IP4

  • Use reciprocal IP with antibodies against suspected interaction partners

  • Include RNase treatment controls to distinguish RNA-mediated from direct protein interactions

This methodology enables reliable isolation of ARL6IP4 protein complexes for interaction studies, enhancing our understanding of its role in splicing regulation networks.

What are common pitfalls when working with ARL6IP4 antibodies and how can they be addressed?

Researchers may encounter several challenges when working with ARL6IP4 antibodies:

• Non-specific bands in Western blots:

  • Increase antibody dilution (1:2000-1:5000) to reduce background

  • Optimize blocking conditions (try 5% BSA instead of milk)

  • Increase washing duration and frequency

  • Pre-adsorb antibody with cell lysate from ARL6IP4-knockout cells

• Weak or no signal:

  • Ensure sample contains adequate ARL6IP4 expression (use positive control lysates)

  • Check antibody compatibility with sample preparation method

  • Optimize epitope retrieval for fixed samples

  • Decrease antibody dilution or increase incubation time

  • Try different antibodies targeting different epitopes of ARL6IP4

• High background in immunofluorescence:

  • Optimize fixation and permeabilization conditions

  • Increase blocking time and concentration

  • Use confocal microscopy for better signal-to-noise ratio

  • Consider signal amplification methods for low-abundance detection

• Cross-reactivity issues:

  • Validate antibody specificity using siRNA knockdown or CRISPR knockout controls

  • Use peptide competition assays with the immunizing peptide

  • Select antibodies with validation in your species of interest

Addressing these common issues improves experimental outcomes and data reliability when working with ARL6IP4 antibodies.

How can researchers validate ARL6IP4 antibody specificity for their particular experimental system?

Comprehensive validation of ARL6IP4 antibody specificity should include:

• Genetic validation approaches:

  • siRNA or shRNA knockdown of ARL6IP4 followed by Western blot or immunofluorescence

  • CRISPR/Cas9 knockout of ARL6IP4 for complete absence of signal

  • Overexpression of tagged ARL6IP4 and detection with both tag-specific and ARL6IP4 antibodies

• Biochemical validation:

  • Peptide competition assays using the immunizing peptide sequence

  • Compare multiple antibodies targeting different epitopes of ARL6IP4

  • Mass spectrometry confirmation of immunoprecipitated protein

• Application-specific validation:

  • For Western blotting: Confirm expected molecular weight (42-45 kDa)

  • For immunofluorescence: Verify expected subcellular localization (nuclear speckles)

  • For immunohistochemistry: Compare with RNA expression data (e.g., from public databases)

• Cross-species validation:

  • When using antibodies across species, verify percent homology of the epitope sequence

  • ABIN2790727 shows predicted reactivity of Cow: 86%, Dog: 86%, Horse: 86%, Human: 100%, Pig: 86%, Rat: 80%

Documentation of these validation steps significantly enhances experimental reproducibility and confidence in results involving ARL6IP4 detection.

What strategies can improve detection of low-abundance ARL6IP4 in different experimental contexts?

For enhanced detection of low-abundance ARL6IP4:

• Sample enrichment techniques:

  • Perform subcellular fractionation to concentrate nuclear proteins

  • Use immunoprecipitation to enrich ARL6IP4 before Western blotting

  • Employ nuclear extraction protocols optimized for splicing factors

• Signal amplification approaches:

  • For Western blotting: Use high-sensitivity ECL or fluorescent detection systems

  • For immunofluorescence: Employ tyramide signal amplification (TSA)

  • Consider biotin-streptavidin amplification systems

• Instrumentation optimization:

  • Use sensitive detection instruments (e.g., cooled CCD cameras for Western blot imaging)

  • Employ confocal or super-resolution microscopy for detailed localization studies

  • Optimize exposure settings to enhance signal without background amplification

• Protocol modifications:

  • Extend primary antibody incubation time (overnight at 4°C or longer)

  • Reduce washing stringency slightly to preserve weak signals

  • Use signal enhancers specific to your detection system

• Antibody selection:

  • Choose polyclonal antibodies for greater epitope recognition and increased sensitivity

  • Select antibodies validated specifically for low-abundance detection

  • Consider using antibody cocktails targeting multiple epitopes simultaneously

These strategies substantially improve detection sensitivity while maintaining specificity, enabling analysis of ARL6IP4 in tissues or conditions with naturally low expression levels.

How can researchers accurately quantify ARL6IP4 expression levels across different experimental conditions?

For reliable quantification of ARL6IP4 expression:

• Western blot quantification:

  • Use equally loaded protein amounts (verified by total protein staining)

  • Include housekeeping proteins as loading controls (β-actin, GAPDH)

  • Capture images within linear dynamic range of detection

  • Use densitometry software with background subtraction

  • Normalize ARL6IP4 signal to loading controls or total protein

• Immunofluorescence quantification:

  • Maintain consistent acquisition parameters across all samples

  • Measure mean fluorescence intensity within defined regions of interest

  • Perform z-stack imaging for complete signal capture

  • Use automated analysis software to minimize subjective interpretation

  • Include calibration controls for fluorescence intensity standardization

• ELISA approaches:

  • Generate standard curves using recombinant ARL6IP4 protein

  • Ensure samples fall within the linear range of the standard curve

  • Run technical and biological replicates for statistical validation

  • Normalize to total protein concentration in samples

• qPCR correlation:

  • Correlate protein levels with mRNA expression for comprehensive analysis

  • Design primers spanning exon-exon junctions to ensure specificity

  • Use multiple reference genes for normalization

What are the appropriate experimental designs for studying ARL6IP4's role in pre-mRNA splicing regulation?

To investigate ARL6IP4's role in splicing regulation, researchers should consider:

• Functional perturbation approaches:

  • siRNA/shRNA knockdown of ARL6IP4 followed by RNA-seq to identify altered splicing events

  • CRISPR/Cas9 knockout for complete functional elimination

  • Expression of dominant-negative ARL6IP4 mutants targeting specific functional domains

  • Rescue experiments with wild-type and mutant ARL6IP4 variants

• Interaction mapping:

  • Co-immunoprecipitation with antibodies against ARL6IP4 followed by mass spectrometry

  • Proximity ligation assays to detect interactions with other splicing factors in situ

  • Use domain-specific antibodies to map interaction interfaces

  • CLIP-seq (Cross-linking immunoprecipitation and sequencing) to identify RNA binding targets

• Splicing assays:

  • Minigene splicing reporters containing exons regulated by ARL6IP4

  • In vitro splicing assays with recombinant ARL6IP4 and nuclear extracts

  • RT-PCR analysis of endogenous alternative splicing events

  • RNA-seq with computational analysis of alternative splicing patterns

• Dynamic analyses:

  • Live-cell imaging with fluorescently tagged ARL6IP4 to track dynamics

  • FRAP (Fluorescence Recovery After Photobleaching) to assess mobility in nuclear speckles

  • Stress-response experiments to evaluate splicing regulation under different cellular conditions

These experimental approaches provide complementary insights into ARL6IP4's mechanistic role in pre-mRNA splicing regulation as demonstrated in the Journal of Cellular Biochemistry publication on SRrp37 .

How should researchers interpret differences in ARL6IP4 localization patterns observed with different antibodies?

When interpreting varying localization patterns detected by different ARL6IP4 antibodies:

• Epitope accessibility considerations:

  • Different fixation methods may expose or mask specific epitopes

  • Protein-protein interactions may block certain epitopes in specific subcellular compartments

  • Post-translational modifications may affect epitope recognition in certain locations

• Isoform-specific detection:

  • Antibodies targeting different regions may recognize distinct isoforms with different localization patterns

  • Compare results with isoform-specific PCR to correlate expression with localization

  • Use antibodies against different regions to build a comprehensive localization map

• Validation approaches:

  • Perform co-localization studies with established nuclear speckle markers

  • Use fluorescently tagged ARL6IP4 expression to confirm antibody-based observations

  • Employ super-resolution microscopy for detailed localization analysis

  • Consider cellular state and cell cycle position when interpreting localization

• Quantitative assessment:

  • Measure co-localization coefficients (Pearson's, Mander's) for objective comparison

  • Quantify relative distribution between nuclear speckles, nucleoplasm, and other compartments

  • Analyze multiple cells and experimental replicates for statistical evaluation

Different localization patterns may reflect biological reality rather than technical artifacts, potentially revealing important insights into ARL6IP4 function and regulation in different cellular contexts.

How can ARL6IP4 antibodies be applied in studying disease-related splicing dysregulation?

ARL6IP4 antibodies offer valuable tools for investigating splicing dysregulation in disease contexts:

• Cancer research applications:

  • Immunohistochemical analysis of ARL6IP4 expression in tumor tissue microarrays

  • Correlation of expression/localization with patient outcomes and cancer subtypes

  • Investigation of ARL6IP4-mediated splicing events affecting oncogenes and tumor suppressors

  • Analysis of post-translational modifications in cancer-specific contexts

• Neurological disorder studies:

  • Examination of ARL6IP4 expression in neurodegenerative disease models

  • Investigation of brain region-specific splicing regulation

  • Correlation with disease-associated splicing pattern alterations

  • Co-localization with disease-associated RNA-binding proteins

• Methodological approaches:

  • Combine ARL6IP4 antibodies with patient-derived samples or disease models

  • Use domain-specific antibodies to detect disease-associated conformational changes

  • Develop phospho-specific antibodies to investigate disease-related post-translational modifications

  • Employ multiplexed imaging to place ARL6IP4 in disease-specific protein interaction networks

• Therapeutic relevance:

  • Monitor changes in ARL6IP4 expression/localization in response to splicing-modulating therapies

  • Evaluate ARL6IP4 as a potential biomarker for diseases with splicing dysregulation

  • Use antibodies to screen for compounds that modulate ARL6IP4 function or interactions

These approaches connect fundamental research on ARL6IP4 to clinically relevant questions about splicing regulation in disease pathogenesis and potential therapeutic interventions.

What methodological advances are improving the application of ARL6IP4 antibodies in current research?

Recent methodological advances enhancing ARL6IP4 antibody applications include:

• Advanced imaging technologies:

  • Super-resolution microscopy (STORM, PALM, SIM) for detailed nuclear speckle organization

  • Live-cell imaging with antibody fragments for dynamic studies

  • Cryo-electron microscopy combined with immuno-gold labeling for structural context

  • Light sheet microscopy for 3D tissue-level analysis of ARL6IP4 distribution

• Single-cell approaches:

  • Single-cell Western blotting for cellular heterogeneity analysis

  • Mass cytometry (CyTOF) with metal-conjugated antibodies for high-dimensional analysis

  • Integration with single-cell transcriptomics to correlate protein with RNA expression/splicing

• Proximity-based methods:

  • BioID or APEX2 proximity labeling combined with ARL6IP4 antibodies

  • Enzyme-mediated proximity labeling to identify context-specific interaction partners

  • Split-BioID approaches to study conditional interactions in specific subcellular locations

• Antibody engineering:

  • Recombinant antibody fragments with enhanced penetration into nuclear structures

  • Bi-specific antibodies for co-detection of ARL6IP4 and interaction partners

  • Nanobodies with reduced size for improved access to dense nuclear regions

These technological advances provide unprecedented insights into ARL6IP4 biology, enabling more sophisticated studies of its dynamic behavior and regulatory functions in splicing regulation contexts.