RABGAP1L Antibody

What is RABGAP1L and what are its primary functions in cellular biology?

RABGAP1L (RAB GTPase Activating Protein 1-Like), also known as TBC1D18, is a member of the Tre2/Bub2/Cdc16 (TBC)-domain-containing protein family involved in the regulation of small membrane-bound GTPases. It primarily functions as a regulator of membrane trafficking through its GAP (GTPase-activating protein) activity toward specific Rab GTPases. RABGAP1L plays critical roles in several cellular processes, including endosomal sorting, maturation, and trafficking. The protein contains a catalytically active TBC domain that promotes GTPase activity and has been demonstrated to regulate various Rab proteins, particularly Rab11A, Rab7A, and Rab10 . Recent studies have shown that RABGAP1L also contributes to cell-autonomous immunity and participates in the elimination of intracellular pathogens by modulating membrane trafficking dynamics . Additionally, RABGAP1L can interact with β1 integrins through its PTB domain to regulate their recycling from endosomes to the plasma membrane, supporting cell migration on 2D surfaces and cell invasion .

How many isoforms of RABGAP1L exist and how do they differ functionally?

Multiple RABGAP1L isoforms have been identified, with research confirming at least four common variants that match the predicted molecular masses of isoforms A, G, H, and I. These isoforms differ primarily in their structural composition, particularly regarding C-terminal extensions. Functional studies have demonstrated that the shorter RABGAP1L isoforms (A and H), which lack C-terminal extensions, exhibit stronger IFN-dependent antiviral effects against influenza A virus compared to other isoforms. Isoform A appears to be the dominantly expressed variant in most cell types tested . The differential expression and activity of these isoforms may be tissue-specific and context-dependent, allowing for specialized functions in different cellular environments. When designing experiments with RABGAP1L antibodies, researchers should consider which isoform(s) they intend to target, as antibody specificity for particular isoforms can significantly impact experimental outcomes and interpretation .

What critical factors should be considered when selecting a RABGAP1L antibody for specific applications?

When selecting a RABGAP1L antibody, researchers should consider:

• Target Epitope Specificity: Determine whether the antibody recognizes specific regions or isoforms of RABGAP1L. For example, some antibodies target the C-terminal region (aa 700 to C-terminus) , while others target specific isoforms like Isoform 10 (AA 11-262) or other regions.

• Application Compatibility: Verify validation data for your specific application (WB, IF/ICC, IHC, IP, ELISA). For instance, antibody catalog #13894-1-AP has been validated for WB (1:500-1:2000), IP (0.5-4.0 μg), IHC (1:20-1:200), and IF/ICC (1:10-1:100) .

• Species Reactivity: Confirm reactivity with your experimental species. Some RABGAP1L antibodies react with human, mouse, and rat samples , while others may have limited cross-reactivity.

• Clonality Selection: Choose between monoclonal (consistent but epitope-restricted) and polyclonal (broader epitope recognition but batch variation) based on your experimental needs. For highly specific isoform detection, monoclonal antibodies may be preferred .

• Validation Data Quality: Review published applications with knockout/knockdown controls, which provide strong evidence for specificity. Publication records indicate successfully validated antibodies for KD/KO, WB, IHC, and IF applications .

What validation methods should be employed to confirm RABGAP1L antibody specificity?

A comprehensive validation approach for RABGAP1L antibodies should include:

• Genetic Controls: Utilize RABGAP1L knockdown (siRNA) or knockout (CRISPR/Cas9) samples as negative controls. Research has validated antibody specificity using siRNA targeting RABGAP1L, confirming specificity through diminished signal in knockdown cells .

• Multiple Antibody Validation: Compare results from different antibodies targeting distinct epitopes of RABGAP1L. Agreement between antibodies targeting different regions (e.g., N-terminal vs. C-terminal) increases confidence in specificity.

• Recombinant Protein Controls: Use purified recombinant RABGAP1L variants as positive controls. For instance, GFP-tagged human RABGAP1L variants with mutations in the PTB domain (F243A, F217A) can confirm epitope recognition patterns .

• Immunoprecipitation-Mass Spectrometry: Confirm antibody pull-down specificity through unbiased protein identification, especially important when studying novel interactions or isoforms.

• Orthogonal Method Correlation: Correlate antibody-based detection with mRNA expression or overexpression systems. Studies have validated antibody specificity by showing consistent results between antibody detection and overexpression of RABGAP1L isoforms A, G, H, and I .

What are the optimal protocols for detecting RABGAP1L in Western blot applications?

For optimal Western blot detection of RABGAP1L:

• Sample Preparation:

  • Use whole cell lysates from appropriate cell lines (validated in HeLa, MCF-7, IMR32 cells)

  • For tissue samples, mouse brain lysates have shown good results

  • Include protease inhibitors to prevent degradation during lysis

• Gel Selection and Transfer:

  • Use 7.5% SDS-PAGE gels due to the large size of RABGAP1L (predicted: 121 kDa)

  • Employ wet transfer systems with extended transfer times (2-3 hours) for complete transfer of high molecular weight proteins

• Antibody Dilution and Incubation:

  • Primary antibody: Use at 1:500-1:2000 dilution for polyclonal (e.g., #13894-1-AP) or 1:5000-1:10000 for high-affinity antibodies (e.g., ab153992)

  • Extended primary antibody incubation at 4°C overnight improves signal quality

  • Secondary antibody: 1:5000-1:10000 HRP-conjugated antibodies for 1 hour at room temperature

• Detection and Analysis:

  • Expected molecular weights range from 93-105 kDa (observed) to 121 kDa (predicted)

  • Multiple bands may represent different isoforms (e.g., 100 kDa, 140 kDa for certain antibodies)

  • If working with overexpressed constructs (e.g., TurboID-V5-tagged RABGAP1L), adjust size expectations accordingly

How should immunofluorescence experiments be optimized for RABGAP1L subcellular localization studies?

For optimal immunofluorescence detection of RABGAP1L localization:

• Fixation and Permeabilization:

  • Test both paraformaldehyde (4%, 15 min) and methanol (-20°C, 10 min) fixation methods, as RABGAP1L associates with membrane structures that may require different fixation approaches

  • For permeabilization, use 0.1-0.2% Triton X-100 for 10 minutes or 0.5% saponin if studying endosomal structures

• Blocking and Antibody Incubation:

  • Block with 5% normal serum from the same species as the secondary antibody for 1 hour

  • Dilute primary antibody between 1:10-1:100 for polyclonal antibodies

  • Incubate overnight at 4°C for optimal signal-to-noise ratio

• Co-labeling Strategy:

  • Include endosomal markers for colocalization studies (Rab11A for recycling endosomes, Rab7A for late endosomes)

  • TurboID-V5-tagged RABGAP1L constructs have been validated for localization studies

  • Consider co-staining with β1 integrin antibodies to study RABGAP1L-integrin interactions

• Imaging Parameters:

  • Use confocal microscopy with Z-stack acquisition to properly resolve endosomal structures

  • Super-resolution techniques (STED, SIM) may be necessary to distinguish between closely associated endosomal compartments

  • Include appropriate controls to determine threshold settings for colocalization analysis

How can RABGAP1L antibodies be utilized to study its role in viral restriction and cell-autonomous immunity?

To investigate RABGAP1L's role in viral restriction and immunity:

• Viral Infection Models:

  • Establish infection models with reporter viruses (e.g., WSN/33-Renilla IAV)

  • Compare viral replication in cells with normal, depleted, or overexpressed RABGAP1L levels

  • Measure viral titers, reporter activity, or viral protein expression at multiple time points

• RABGAP1L Manipulation Approaches:

  • Use siRNA panels targeting different RABGAP1L regions (validated with 5 different siRNAs)

  • Generate stable cell lines overexpressing specific RABGAP1L isoforms (A, G, H, I)

  • Create RABGAP1L mutants (R612A, Q621A, R612A/Q621A, KK784EE) to study domain-specific functions

• Interferon Response Analysis:

  • Pre-treat cells with IFNα2 before infection to study RABGAP1L's role in IFN-mediated antiviral activity

  • Analyze ISG expression patterns in RABGAP1L-manipulated cells using qRT-PCR or RNA-seq

  • Assess phosphorylation status of STAT1/STAT2 to determine if RABGAP1L affects IFN signaling

• Endosomal Trafficking Visualization:

  • Use live-cell imaging with fluorescently tagged RABGAP1L and viral particles

  • Apply endosomal markers to track virus-containing compartments

  • Implement proximity labeling techniques using TurboID-tagged RABGAP1L to identify interacting partners during infection

What experimental approaches can reveal RABGAP1L's interactions with Rab GTPases in membrane trafficking?

To elucidate RABGAP1L-Rab GTPase interactions:

• GAP Activity Assays:

  • Perform in vitro GAP activity assays using purified RABGAP1L (or TBC domain) and various Rab GTPases

  • Measure GTP hydrolysis rates to determine specificity toward different Rab proteins (Rab7A, Rab10, Rab11A)

  • Compare wild-type and catalytically inactive mutants (R612A) to confirm GAP-dependent effects

• Rab Activation Status Monitoring:

  • Use GST-effector pull-down assays to measure GTP-bound (active) Rab levels in cells with manipulated RABGAP1L expression

  • Apply FRET-based biosensors to visualize Rab activation dynamics in real-time

  • Implement dominant-negative Rab constructs (e.g., dominant-negative Rab11a) to dissect pathway specificity

• Proximity-Based Interaction Mapping:

  • Apply proximity labeling approaches with TurboID-tagged RABGAP1L to identify proximal interactors in living cells

  • Combine with mass spectrometry to generate comprehensive interactomes

  • Validate key interactions through co-immunoprecipitation using RABGAP1L antibodies

• Endosomal Trafficking Analysis:

  • Track recycling kinetics of fluorescently labeled cargo proteins (e.g., β1 integrins) in RABGAP1L-manipulated cells

  • Measure endosomal maturation rates using pH-sensitive dyes or ratiometric imaging

  • Quantify colocalization between RABGAP1L, various Rab proteins, and cargo molecules throughout the endocytic pathway

• Structural Domain Contribution:

  • Generate RABGAP1L constructs with mutations in key domains (TBC domain, PTB domain) to dissect their contributions

  • Analyze the effects of these mutations on Rab binding, GAP activity, and functional outcomes in trafficking assays

  • Compare the impact of different isoforms on specific Rab GTPase regulations

How should researchers address discrepancies in RABGAP1L molecular weight detection in Western blot analyses?

When encountering molecular weight discrepancies:

• Isoform Variation Assessment:

  • RABGAP1L has multiple isoforms with predicted weights ranging from 93 kDa to 140 kDa

  • Different antibodies target different epitopes, potentially detecting distinct isoform subsets

  • Compare observed bands with predicted weights of known isoforms (A, G, H, I and others)

• Post-translational Modification Analysis:

  • Treatment with phosphatases or deglycosylation enzymes can reveal whether PTMs contribute to increased molecular weight

  • Mass spectrometry analysis of immunoprecipitated RABGAP1L can identify specific modifications

  • Compare migration patterns across different cell types and conditions to identify context-dependent modifications

• Sample Preparation Optimization:

  • Ensure complete denaturation by increasing SDS concentration or boiling time

  • Test different lysis buffers to preserve protein integrity

  • Include fresh protease inhibitors to prevent degradation that may result in lower molecular weight bands

• Antibody Validation with Controls:

  • Use lysates from cells overexpressing specific RABGAP1L isoforms as positive controls

  • Include RABGAP1L knockdown/knockout samples as negative controls

  • Test multiple RABGAP1L antibodies targeting different epitopes to confirm band specificity

• Resolution Improvement:

  • Use gradient gels (4-15%) for better separation of high molecular weight proteins

  • Extend running time to improve resolution between closely migrating bands

  • Consider Phos-tag or other specialty gels if phosphorylation contributes to migration differences

What strategies can address weak or inconsistent RABGAP1L detection in immunohistochemistry applications?

For improving inconsistent IHC detection:

• Antigen Retrieval Optimization:

  • Test multiple antigen retrieval methods: TE buffer pH 9.0 has been suggested for optimal results, with citrate buffer pH 6.0 as an alternative

  • Adjust retrieval time and temperature (microwave, pressure cooker, water bath) to find optimal conditions

  • For formalin-fixed tissues, extend retrieval times to ensure complete epitope unmasking

• Antibody Concentration Adjustment:

  • Perform titration experiments across a wide dilution range (1:20-1:200 has been recommended for some antibodies)

  • Consider signal amplification systems (polymer-HRP, TSA) for low-abundance detection

  • Test longer primary antibody incubation times (overnight at 4°C versus 1-2 hours at room temperature)

• Tissue Processing Considerations:

  • Freshly prepared sections often yield better results than stored slides

  • Minimize fixation time in formalin when possible for prospective studies

  • Test frozen versus paraffin-embedded sections if fixation affects epitope recognition

• Blocking Optimization:

  • Increase blocking agent concentration (5-10% normal serum)

  • Include additional blocking steps for endogenous peroxidase, biotin, or non-specific binding

  • Test commercial blocking solutions specifically designed for difficult antibodies

• Positive Control Selection:

  • Include tissues with known RABGAP1L expression (human hepatocirrhosis tissue, human placenta tissue have shown positive results)

  • Consider artificially overexpressing RABGAP1L in control samples when studying tissues with low endogenous expression

  • Use multiple antibodies against different RABGAP1L epitopes to confirm staining patterns

How can researchers utilize RABGAP1L antibodies to investigate its role in Alzheimer's disease-related APP trafficking?

Recent research suggests RABGAP1L may interact with amyloid precursor protein (APP) through its PTB domain and the YENPTY motif in APP's cytosolic tail . To investigate this relationship:

• Interaction Verification Methods:

  • Perform co-immunoprecipitation using RABGAP1L antibodies to pull down endogenous APP

  • Apply proximity ligation assays (PLA) to visualize and quantify RABGAP1L-APP interactions in situ

  • Use structural modeling approaches (AlphaFold3) to predict interaction interfaces and design validation experiments

• APP Trafficking Analysis:

  • Track fluorescently tagged APP in neuronal cells with manipulated RABGAP1L levels

  • Quantify endosomal sorting and processing of APP using subcellular fractionation followed by Western blotting

  • Compare APP processing (measure Aβ peptides by ELISA) in cells with normal versus depleted RABGAP1L

• Domain-Specific Contributions:

  • Generate PTB domain mutants of RABGAP1L to disrupt APP binding while maintaining other functions

  • Compare wild-type and GAP-inactive (R612A) RABGAP1L to determine if GAP activity is required for APP trafficking effects

  • Investigate if RABGAP1L-dependent regulation of APP trafficking differs from its effects on other NPXY-containing cargo

• Neuronal Model Systems:

  • Compare findings between human and rodent neurons to identify conserved mechanisms

  • Apply RABGAP1L antibodies in brain tissue sections from Alzheimer's disease models to assess expression patterns

  • Utilize iPSC-derived neurons from Alzheimer's patients to study disease-relevant alterations in RABGAP1L-APP interactions