ARL6IP1 Antibody

What is ARL6IP1 and what is its structural organization?

ARL6IP1 (ADP-Ribosylation Factor-Like 6 Interacting Protein 1) is a transmembrane protein with a distinctive structural arrangement. Research has revealed that ARL6IP1 is a three-spanning transmembrane protein with both the amino and C-termini facing the cytoplasm . Its structure includes two long hydrophobic regions (transmembrane helical hairpins TM1+2 and TM3+4) separated by an accessible linker segment . Advanced structural modeling using AlphaFold predicts two membrane-embedded helical hairpins with two amphipathic helices . This RHD-like (reticulon homology domain-like) structural arrangement is critical for its function in membrane shaping and cellular homeostasis.

What are the key functional domains of ARL6IP1 that can be targeted by antibodies?

ARL6IP1 contains several distinct regions that can be targeted by specific antibodies:

• C-terminal region: Critical for protein-protein interactions and often targeted by antibodies like ABIN6990462

• Middle region: Contains the sequence "GVSCFVMFLC LADYLVPILA PRIFGSNKWT TEQQQRFHEI CSNLVKTRRR" which is highly conserved across species and targeted by antibodies like ABIN2788828

• N-terminal region: Contains putative LC3-interacting regions (LIRs)

• Cytoplasmic loop between RHDs: Contains recognition sites for some antibodies

Understanding these domains helps in selecting antibodies that target specific functional regions for various experimental applications.

What species reactivity should be considered when selecting an ARL6IP1 antibody?

When selecting an ARL6IP1 antibody, researchers should consider cross-species reactivity based on experimental models. ARL6IP1 is highly conserved across species, with many antibodies demonstrating multi-species reactivity. The antibody ABIN2788828, for example, shows predicted reactivity with: Human (100%), Mouse (100%), Rat (100%), Cow (100%), Dog (100%), Goat (93%), Guinea Pig (100%), Horse (100%), Rabbit (100%), Sheep (100%), and Zebrafish (100%) . For studies involving less common model organisms, it's advisable to verify sequence homology in the targeted epitope region. For human-specific research, several antibodies like USBI123534 are available with specific human reactivity .

What are the optimal applications for ARL6IP1 antibodies in cellular localization studies?

For cellular localization studies of ARL6IP1, immunofluorescence (IF) techniques using antibodies like ABIN6990462 are particularly effective . When designing these experiments:

• Use antibodies validated for IF applications that target accessible epitopes (C-terminal or middle regions)

• Implement dual labeling with organelle markers, particularly for endoplasmic reticulum (ER) and mitochondria-associated membranes (MAMs) where ARL6IP1 localizes

• Consider fluorescence protease protection assays to confirm the topology of ARL6IP1 within membranes

• For co-localization studies with interacting partners like FXR1, use antibodies that don't interfere with protein-protein interaction sites

These approaches can verify ARL6IP1's subcellular distribution and clarify its role in maintaining ER-mitochondrial homeostasis.

How can ARL6IP1 antibodies be employed in protein-protein interaction studies?

ARL6IP1 antibodies are valuable tools for investigating protein-protein interactions through several methodological approaches:

• Co-immunoprecipitation (Co-IP): Use ARL6IP1 antibodies like ABIN6990462 to pull down ARL6IP1 and its interacting partners such as FXR1/FXR2 . Western blotting with antibodies against potential binding partners can then confirm interactions.

• Proximity ligation assays: Combine ARL6IP1 antibodies with antibodies against suspected interacting proteins to visualize protein complexes in situ.

• FRET/BRET analysis: Use ARL6IP1 antibodies for validation of energy transfer experiments examining dynamic protein interactions.

• Immunofluorescence co-localization: Employ ARL6IP1 antibodies alongside antibodies for proteins like FAM134B or AMFR to examine spatial relationships .

When performing these studies, it's critical to select antibodies targeting epitopes that don't interfere with the interaction domains being studied.

What controls and validation steps are essential when using ARL6IP1 antibodies in Western blotting?

When employing ARL6IP1 antibodies for Western blotting, several critical controls and validation steps should be implemented:

• Positive controls: Include lysates from tissues/cells known to express ARL6IP1 (e.g., neuronal cells for endogenous expression or ARL6IP1-transfected cells for overexpression)

• Negative controls: Utilize ARL6IP1 knockout samples when available, or lysates from cells with ARL6IP1 knockdown by siRNA/shRNA

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide to confirm specificity

• Cross-validation: Use multiple antibodies targeting different epitopes of ARL6IP1 to confirm findings

• Molecular weight verification: ARL6IP1 should appear at the expected size; variant forms (like the K193Ffs variant) may show slightly higher molecular weight

• Loading controls: Include appropriate housekeeping proteins while avoiding those that might be affected by experimental conditions

These validation steps ensure reliable and reproducible results when studying ARL6IP1 expression and modifications.

How can ARL6IP1 antibodies be utilized to study neuroinflammatory processes in disease models?

ARL6IP1 antibodies can be strategically employed to investigate neuroinflammatory processes in various disease models through multiple approaches:

• Immunohistochemical analysis of brain tissue sections using antibodies like ABIN6990462 to evaluate ARL6IP1 expression patterns in relation to neuroinflammatory markers (IBA1, GFAP)

• Western blotting to quantify ARL6IP1 protein levels in neuroinflammatory conditions, particularly in models of hereditary spastic paraplegia where Arl6ip1 knockout mice show significant neuroinflammation

• Dual immunofluorescence staining to examine co-localization of ARL6IP1 with microglial polarization markers (M1 markers: Cxcr3-1, Cd40, and Cd80; M2 markers: Arg-1, Cd163, and Igf-1)

• Correlation analysis between ARL6IP1 expression and levels of proinflammatory cytokines and chemokines in brain tissue samples

Research has demonstrated that Arl6ip1 knockout mice exhibit increased neuroinflammation with significant changes in microglial M1/M2 polarization, making antibody-based detection of ARL6IP1 valuable for monitoring therapeutic interventions targeting this pathway .

What methodological considerations are important when studying ARL6IP1's role in Alzheimer's disease pathology?

When investigating ARL6IP1's role in Alzheimer's disease (AD) pathology, several methodological considerations are essential:

• Combined detection approaches: Use ARL6IP1 antibodies alongside BACE1 and amyloid-β antibodies to establish correlations between ARL6IP1 expression and AD pathological markers

• Translational regulation analysis: Implement ARL6IP1 antibodies in RNA immunoprecipitation (RIP) assays to study its interaction with FXR1 and the 5'UTR of BACE1 mRNA

• Intervention validation: When testing compounds like conophylline (CNP) that target ARL6IP1, use antibodies to verify target engagement through co-immunoprecipitation with interacting partners

• Microscopy techniques: Employ high-resolution microscopy with ARL6IP1 antibodies to visualize its colocalization with RNA-protein complexes using RNA tracking systems like MS2-MCP

• In vivo validation: Utilize ARL6IP1 antibodies to confirm the efficacy of AAV-mediated ARL6IP1 knockdown in APP/PS1 mice and subsequent effects on amyloidogenesis

These methodological approaches can reveal how ARL6IP1 mediates BACE1 translation through FXR1-dependent mechanisms, providing insights into potential therapeutic targets for AD.

How can ARL6IP1 antibodies contribute to gene therapy development for hereditary spastic paraplegia?

ARL6IP1 antibodies play a crucial role in developing and validating gene therapy approaches for hereditary spastic paraplegia (HSP) through several methodological applications:

• Target validation: Use antibodies to confirm the absence or mutation of ARL6IP1 in patient samples, such as in cases with the c.577–580delAAAC variant that represents a knockout allele

• Therapeutic delivery confirmation: Employ antibodies to verify successful AAV9-ARL6IP1 delivery in animal models, demonstrating restored protein expression in target tissues

• Phenotype correlation: Use immunohistochemistry with ARL6IP1 antibodies to correlate protein expression with improvements in limb paraplegia and gait abnormalities in mouse models

• Mechanistic studies: Apply antibodies to investigate how restored ARL6IP1 expression alleviates demyelination of axons and neuroinflammation in the white matter, including the corticospinal tract

• Cellular response monitoring: Use antibodies to track how ARL6IP1 restoration affects mitochondrial dysfunction and dysregulated autophagy in neuronal cells

These approaches contribute to establishing ARL6IP1 as a viable target for HSP gene therapy, with antibodies serving as critical tools for validation and mechanism elucidation.

How can researchers investigate ARL6IP1's role in mitochondria-associated membrane (MAM) function?

To investigate ARL6IP1's role in mitochondria-associated membrane (MAM) function, researchers can employ several antibody-based methodological approaches:

• Subcellular fractionation with immunoblotting: Use ARL6IP1 antibodies to detect its presence in isolated MAM fractions compared to pure ER or mitochondrial fractions

• Proximity-based labeling: Combine ARL6IP1 antibodies with BioID or APEX2 proximity labeling to identify proteins in close spatial proximity at MAMs

• Super-resolution microscopy: Employ ARL6IP1 antibodies with structured illumination or STORM microscopy to visualize its precise localization at ER-mitochondria contact sites

• Co-immunoprecipitation studies: Use ARL6IP1 antibodies to identify interactions with known MAM proteins like LC3B and BCl2L13

• Functional assays: After manipulating ARL6IP1 levels, use antibodies to correlate its expression with calcium transfer, lipid exchange, or autophagosome formation at MAMs

Research has established that ARL6IP1 plays a crucial role in connecting the endoplasmic reticulum and mitochondria as a member of MAMs, maintaining organelle homeostasis through direct interactions with autophagy-related proteins .

What techniques can be used to study ARL6IP1 ubiquitination patterns using specific antibodies?

To investigate ARL6IP1 ubiquitination patterns, researchers can implement several specialized techniques:

• Immunoprecipitation with ubiquitin analysis:

  • Pull down ARL6IP1 using specific antibodies like ABIN6990462

  • Probe with anti-ubiquitin antibodies to detect ubiquitinated forms

  • Verify with reciprocal IP using anti-ubiquitin antibodies followed by ARL6IP1 detection

• Site-specific ubiquitination mapping:

  • Use mass spectrometry following ARL6IP1 immunoprecipitation to identify specific lysine residues that undergo ubiquitination

  • Focus on K96 and K114, which have been identified as ubiquitination sites by AMFR

• Co-expression studies:

  • Express V1-ARL6IP1 with AMFR-V2 or catalytically inactive AMFR RINGmut-V2

  • Use antibodies to detect ubiquitinated ARL6IP1 and quantify the ratio of peptides ubiquitinated at specific lysine residues

• Stress-induced ubiquitination:

  • Induce ER stress in cellular models

  • Use ARL6IP1 antibodies to detect endogenous ubiquitinated forms under stress conditions

• In vitro ubiquitination assays:

  • Combine purified components (E1, E2, AMFR E3 ligase, ubiquitin, and ARL6IP1)

  • Use antibodies to verify that ARL6IP1-K96 is ubiquitinated by AMFR in vitro

These methodologies can reveal how ubiquitination regulates ARL6IP1 function and interactions with other proteins, particularly in ER-stress responses.

How can researchers investigate ARL6IP1's interaction with RNA-binding proteins in translational control?

To investigate ARL6IP1's interaction with RNA-binding proteins in translational control, researchers can implement several advanced methodological approaches:

• RNA immunoprecipitation (RIP) combined with RT-qPCR:

  • Use ARL6IP1 antibodies to pull down protein-RNA complexes

  • Quantify associated mRNAs, particularly BACE1 mRNA with its 5'UTR

  • Compare results with and without treatments like conophylline (CNP)

• Co-immunoprecipitation with RNA-binding proteins:

  • Use ARL6IP1 antibodies to immunoprecipitate the protein complex

  • Probe for RNA-binding proteins like FXR1/FXR2 in the precipitated fraction

  • Perform reciprocal IP with FXR1/FXR2 antibodies and detect ARL6IP1

• Visualization of protein-RNA interactions:

  • Employ MS2-MCP RNA tracking systems to visualize the 5'UTR

  • Use immunofluorescence to detect colocalization of ARL6IP1, FXR1, and the labeled RNA

  • Quantify changes in colocalization upon treatments that affect this interaction

• RNA pulldown with mass spectrometry:

  • Label the 5'UTR with 5-bromo-UTP (BrU)

  • Use anti-BrU antibody for RNA pulldown

  • Identify associated proteins by LC-MS/MS

  • Validate ARL6IP1's presence in the complex

• Functional translation assays:

  • Utilize 5'UTR-luciferase reporter systems

  • Manipulate ARL6IP1 levels through knockdown or overexpression

  • Measure changes in translational efficiency

  • Correlate with ARL6IP1-FXR1 interaction status

These approaches can reveal how ARL6IP1 mediates the effect of small molecules like CNP on BACE1 translation through dynamic interactions with FXR1 and the 5'UTR.

What criteria should guide the selection of appropriate ARL6IP1 antibodies for specific research applications?

When selecting ARL6IP1 antibodies for specific research applications, researchers should consider these methodological criteria:

• Epitope specificity and accessibility:

  • For Western blotting: Antibodies targeting the C-terminus (ABIN6990462) or middle region (ABIN2788828) perform well

  • For immunoprecipitation: Choose antibodies recognizing accessible epitopes in the native protein conformation

  • For immunohistochemistry: Select antibodies validated for IHC(p) applications like ABIN6990462

• Host species compatibility:

  • Consider experimental design requirements for multi-labeling

  • Mouse monoclonal antibodies (USBI123534) for co-staining with rabbit antibodies

  • Rabbit polyclonal antibodies when maximum sensitivity is required

• Application-specific validation:

  • Verify that the antibody has been validated for your specific application (WB, ELISA, IF, IHC)

  • Review literature where the antibody has been used successfully

• Species cross-reactivity:

  • For comparative studies: Choose antibodies with broad cross-reactivity (ABIN2788828)

  • For human-specific research: Select antibodies with validated human reactivity

• Clonality considerations:

  • Polyclonal antibodies offer higher sensitivity by recognizing multiple epitopes

  • Monoclonal antibodies provide greater specificity for a single epitope

These selection criteria ensure optimal antibody performance for the specific experimental context and research objectives.

How can researchers troubleshoot non-specific binding when using ARL6IP1 antibodies?

When encountering non-specific binding with ARL6IP1 antibodies, researchers can implement these methodological troubleshooting steps:

• Optimization of blocking conditions:

  • Test alternative blocking agents (BSA, casein, non-fat dry milk)

  • Increase blocking time and concentration

  • Add detergents like Tween-20 to reduce hydrophobic interactions

• Antibody dilution optimization:

  • Perform titration experiments to determine optimal antibody concentration

  • Generally start with manufacturer recommendations and adjust as needed

  • Higher dilutions typically reduce non-specific binding

• Validation with negative controls:

  • Use ARL6IP1 knockout or knockdown samples as negative controls

  • Implement peptide competition assays with the immunizing peptide

  • Include secondary antibody-only controls to identify non-specific secondary binding

• Sample preparation modifications:

  • For membrane proteins like ARL6IP1, optimize lysis conditions to maintain protein conformation

  • Consider native vs. denaturing conditions based on the antibody's epitope recognition properties

  • Adjust fixation protocols for immunohistochemistry to preserve epitope accessibility

• Cross-validation strategies:

  • Use multiple ARL6IP1 antibodies targeting different epitopes

  • Compare results with antibodies from different manufacturers

  • Confirm findings with alternative detection methods (e.g., mass spectrometry)

These systematic troubleshooting approaches can significantly improve signal specificity when working with ARL6IP1 antibodies across various experimental applications.

What are the best practices for preserving ARL6IP1 epitopes during sample preparation for immunohistochemistry?

When preparing samples for immunohistochemical detection of ARL6IP1, researchers should implement these methodological best practices to preserve epitopes:

• Fixation protocol optimization:

  • For ARL6IP1 as a transmembrane protein, use mild fixation with 2-4% paraformaldehyde

  • Limit fixation time to 12-24 hours to prevent over-fixation and epitope masking

  • Consider dual fixation with low concentrations of glutaraldehyde (0.1-0.2%) for membrane structure preservation

• Antigen retrieval techniques:

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) is effective for antibodies targeting the C-terminal region

  • For middle region epitopes, test both citrate and EDTA (pH 9.0) buffers to determine optimal conditions

  • Carefully control retrieval times to prevent tissue damage while ensuring adequate epitope exposure

• Permeabilization considerations:

  • As ARL6IP1 spans the membrane with cytoplasmic domains, include adequate permeabilization

  • Use mild detergents like 0.1-0.3% Triton X-100 or 0.05-0.1% saponin

  • Adjust permeabilization based on the antibody's target epitope location

• Section thickness and tissue processing:

  • For paraffin embedding, use standardized processing protocols to minimize protein denaturation

  • Prepare thin sections (4-6 μm) to improve antibody penetration

  • For cryosections, use optimal cutting temperature and rapid freezing to preserve membrane structure

• Blocking strategies:

  • Implement dual blocking with serum matching the secondary antibody host species

  • Add protein blockers like BSA (1-3%) to reduce background

  • Consider specific blockers for endogenous peroxidase, biotin, or Fc receptors when applicable

These optimized sample preparation practices enhance ARL6IP1 epitope preservation and accessibility, resulting in more specific and reproducible immunohistochemical detection.

How can ARL6IP1 antibodies be utilized in high-throughput screening for therapeutic compounds?

ARL6IP1 antibodies can be strategically incorporated into high-throughput screening (HTS) platforms through several methodological approaches:

• Cell-based assay development:

  • Create reporter cell lines expressing ARL6IP1 fused to fluorescent proteins

  • Use ARL6IP1 antibodies to validate proper localization and expression levels

  • Develop immunofluorescence-based assays to screen compounds that alter ARL6IP1 localization or levels

• Target engagement verification:

  • Implement cellular thermal shift assays (CETSA) with ARL6IP1 antibodies to detect compound binding

  • Validate hits from primary screens through co-immunoprecipitation to assess changes in ARL6IP1 interactions with partners like FXR1

  • Use antibodies in Western blotting to quantify downstream effects on proteins like BACE1

• Functional consequence screening:

  • Develop assays measuring ARL6IP1-dependent functions (ER shaping, MAM integrity)

  • Use antibodies to correlate functional changes with ARL6IP1 expression/modification

  • Screen for compounds that rescue defects in ARL6IP1 knockout or mutant models

• Mechanistic pathway analysis:

  • Employ antibodies in high-content screening microscopy to monitor ARL6IP1's interactions with RNA-protein complexes

  • Analyze effects of hit compounds on ARL6IP1 ubiquitination patterns

  • Screen for molecules that modulate ARL6IP1's role in translational control

This methodological framework can identify compounds like conophylline (CNP) that target ARL6IP1-dependent pathways, potentially leading to new therapeutic approaches for conditions like Alzheimer's disease and hereditary spastic paraplegia .

What emerging technologies can enhance the specificity and sensitivity of ARL6IP1 detection in complex samples?

Several cutting-edge technologies can significantly enhance ARL6IP1 detection specificity and sensitivity in complex biological samples:

• Proximity ligation assay (PLA) advancements:

  • Combine ARL6IP1 antibodies with antibodies against interacting partners

  • Generate fluorescent signals only when proteins are in close proximity (<40 nm)

  • This approach provides superior signal-to-noise ratio in tissue samples with high specificity for protein interactions

• Mass cytometry (CyTOF) applications:

  • Label ARL6IP1 antibodies with rare earth metals

  • Achieve highly multiplexed detection without fluorescence spectral overlap issues

  • Enable simultaneous analysis of ARL6IP1 with dozens of other proteins in single cells

• Single-molecule detection methods:

  • Implement stochastic optical reconstruction microscopy (STORM) with ARL6IP1 antibodies

  • Achieve 10-20 nm resolution to precisely localize ARL6IP1 at subcellular structures

  • Visualize individual ARL6IP1 molecules within membrane domains

• Antibody-based proximity biotinylation:

  • Fuse biotin ligase to anti-ARL6IP1 antibody fragments

  • Identify neighboring proteins in living cells without overexpression artifacts

  • Map the ARL6IP1 proximal proteome in different cellular compartments

• Spectral flow cytometry:

  • Utilize unmixing algorithms to separate overlapping fluorophore signals

  • Achieve higher dimensional analysis of ARL6IP1 with multiple markers

  • Quantify subtle changes in ARL6IP1 expression across heterogeneous cell populations

These emerging technologies overcome traditional limitations in antibody-based detection, providing unprecedented insights into ARL6IP1's spatial organization, interactions, and dynamics in complex biological systems.

How can researchers integrate ARL6IP1 antibody-based techniques with multi-omics approaches?

Researchers can implement integrative strategies combining ARL6IP1 antibody-based techniques with multi-omics approaches through these methodological frameworks:

• Antibody-based proteomics integration:

  • Use ARL6IP1 antibodies for immunoprecipitation followed by mass spectrometry

  • Identify post-translational modifications and interacting partners

  • Correlate findings with global proteomics data to place ARL6IP1 in broader pathway contexts

• Spatial transcriptomics correlation:

  • Perform immunofluorescence with ARL6IP1 antibodies on tissue sections

  • Combine with spatial transcriptomics to correlate protein localization with local gene expression profiles

  • Identify region-specific relationships between ARL6IP1 and its transcriptional regulators or targets

• Single-cell multi-modal analysis:

  • Implement CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing)

  • Use oligonucleotide-tagged ARL6IP1 antibodies to simultaneously measure protein and mRNA

  • Reveal cell-type-specific relationships between ARL6IP1 protein levels and transcriptome

• Functional genomics validation:

  • Use CRISPR screens to identify genes affecting ARL6IP1 function

  • Validate hits with ARL6IP1 antibody-based assays measuring protein localization, interaction, or activity

  • Integrate results with transcriptomic and proteomic datasets to build comprehensive pathway models

• RNA-protein interaction mapping:

  • Combine RNA immunoprecipitation using ARL6IP1 antibodies with RNA-seq

  • Identify directly bound transcripts, particularly those with regulatory 5'UTRs like BACE1

  • Integrate with translatomics data to correlate ARL6IP1-RNA interactions with translational efficiency