TBC1D3G Antibody

What is TBC1D3G and what is its biological function?

TBC1D3G (TBC1 Domain Family Member 3G) functions as a GTPase-activating protein (GAP) specifically for RAB5, but does not act on RAB4 or RAB11 . It belongs to the TBC1D3 family, which is characterized by the presence of a RAB-GAP domain called the TBC1 domain . These proteins typically function as attenuators of RABs, which are considered master switches of membrane traffic. TBC1D3G is part of a hominoid-specific gene family that evolved to modulate signaling pathways of higher complexity . The TBC1D3 family has been identified as a hominoid-specific oncogene that can enhance cell proliferation and is encoded by a cluster of eight paralogs on chromosome 17 .

Which applications are most suitable for TBC1D3G antibodies?

TBC1D3G antibodies are primarily validated for the following applications:

The choice of application depends on your experimental question. For protein expression analysis across different samples, Western Blot is typically used. For localization studies, ICC or IHC are preferred methods. ELISA is useful for quantitative protein detection from solution samples .

How should I design positive and negative controls for TBC1D3G antibody experiments?

Proper control selection is critical for valid experimental interpretation when working with TBC1D3G antibodies:

Positive Controls:

• Cell lines known to express TBC1D3G (such as HEK-293 cells)

• Recombinant TBC1D3G protein (available as catalog numbers ABIN7544194 or ABIN3073765)

• Triple-negative breast cancer (TNBC) tissue samples, which show higher expression of TBC1D3G compared to other breast cancer subtypes

Negative Controls:

• Unstained samples to establish autofluorescence baseline

• Fluorescence Minus One (FMO) controls for flow cytometry experiments to determine gating boundaries

• TBC1D3G knockout cell lines using CRISPR-Cas9 or siRNA approaches

• Mouse tissue samples (as TBC1D3 is a hominoid-specific gene)

Avoid using isotype controls as they often have different binding properties and fluorophore-to-antibody ratios compared to your test antibody . For flow cytometry, FMO controls are more reliable for establishing positive signal thresholds.

What are the critical considerations for sample preparation when using TBC1D3G antibodies?

Sample preparation significantly impacts TBC1D3G antibody performance:

How should I analyze flow cytometry data using TBC1D3G antibodies?

When analyzing flow cytometry data with TBC1D3G antibodies, follow these methodological steps:

• Gating Strategy:

  • First gate on live, single cells using appropriate viability dye and forward/side scatter parameters

  • Dead cells soak up antibody non-specifically and can lead to false positives

  • Use doublet discrimination to exclude cell aggregates

• Statistical Analysis:

  • Report percentage of positive cells for frequency analysis

  • Use Median Fluorescence Intensity (MFI) rather than mean for expression level analysis (as flow cytometry data is typically displayed on logarithmic scales)

  • Calculate fold-change in MFI as MFI(sample)/MFI(control) when comparing expression levels between samples

• Visualization:

  • Create overlay plots to compare stained samples with unstained controls

  • For multiparameter analysis, use bivariate plots with appropriate FMO controls to set boundaries

Remember that small changes in negative populations can translate into large changes in fold-difference due to the logarithmic scale, so interpret fold-changes with caution .

What are the potential pitfalls in interpreting TBC1D3G antibody experimental results?

Several challenges exist when interpreting results from TBC1D3G antibody experiments:

• Cross-reactivity: TBC1D3G belongs to a family with several paralogs (including TBC1D3, TBC1D3B, TBC1D3C) that share significant sequence homology . Verify antibody specificity against these related proteins.

• Isoform specificity: Different antibodies may recognize different epitopes and potentially different TBC1D3G isoforms. For example, some antibodies target regions such as amino acids 231-281 or 312-549 .

• Species considerations: TBC1D3 is a hominoid-specific gene, so results from non-primate models must be interpreted with caution . Most commercial antibodies show reactivity to human TBC1D3G .

• Background signals: In immunohistochemistry, autofluorescence can be mistaken for positive staining. Always include proper unstained controls .

• Quantification methods: For Western blot analysis, normalize TBC1D3G signals to appropriate loading controls (such as GAPDH) as demonstrated in previous studies .

How can TBC1D3G antibodies be used to investigate its role in cancer progression and immune responses?

TBC1D3G has been implicated in cancer progression and immune responses, making it an important target for advanced research:

• Cancer Biomarker Studies:

  • TBC1D3G is expressed at higher levels in triple-negative breast cancers (TNBC) compared to other breast cancer subtypes

  • TBC1D3 family can be used as a prognostic biomarker in kidney renal clear cell carcinoma (KIRC)

  • Flow cytometry with TBC1D3G antibodies can identify cells with altered TBC1D3G expression levels in patient-derived samples

• Immune Response Investigation:

  • TBC1D3 family expression correlates with immune checkpoint inhibitors including CD160, CTLA4, CD244, LAG3, PDCD1, and TIGIT

  • TBC1D3G expression is associated with CD4+ T cell infiltration levels in tumor microenvironments

  • Correlation analysis between TBC1D3 expression and marker sets of neutrophils, Th1, Th2, Treg, and T cell exhaustion revealed significant relationships

• Methodology Approaches:

  • Combine TBC1D3G antibody staining with other immune cell markers for multiparameter flow cytometry

  • Use immunoprecipitation with TBC1D3G antibodies followed by mass spectrometry to identify interacting partners

  • Employ tissue microarrays with TBC1D3G antibodies to analyze expression patterns across large numbers of patient samples

What methodological approaches can be used to investigate TBC1D3G's role in membrane trafficking?

TBC1D3G's function as a GTPase activating protein for RAB5 suggests its involvement in membrane trafficking pathways. Several methodological approaches can be used to investigate this role:

• Colocalization Studies:

  • Use TBC1D3G antibodies in combination with markers for early endosomes (EEA1), late endosomes, or recycling endosomes

  • Confocal microscopy with dual immunofluorescence staining can reveal spatial relationships between TBC1D3G and trafficking components

• Functional Assays:

  • Macropinocytosis assays using fluorescent dextran uptake in cells with modulated TBC1D3G expression

  • EGFR trafficking assays, as TBC1D3 has been shown to delay EGFR degradation and extend receptor lifespan

  • In vitro GAP assays using purified components to measure GAP activity toward RAB5

• Protein-Protein Interaction Analysis:

  • Immunoprecipitation with TBC1D3G antibodies to pull down protein complexes

  • GST pull-down assays using GST-fusion proteins of TBC1D3G

  • Proximity ligation assays to visualize TBC1D3G interactions with potential binding partners in situ

• Domain Mapping:

  • Generate deletion mutants (such as TBC1D3-92-319, TBC1D3-2-319, etc.) to map functional domains

  • Site-directed mutagenesis of key residues (e.g., R147A mutations) to investigate catalytic functions

How can TBC1D3G antibodies be used to study its involvement in antibody-dependent enhancement of viral infections?

Recent research has identified TBC1D3G as potentially involved in antibody-dependent enhancement (ADE) of dengue virus (DENV) infection . Methodological approaches to investigate this function include:

• Infection Models:

  • Develop ADE assays using cells expressing TBC1D3G and FcγRIIa

  • Compare binding and internalization of IgG-DENV complexes in control versus TBC1D3G knockout cells

• Mechanistic Studies:

  • Use TBC1D3G antibodies for immunofluorescence to track its localization during viral entry

  • Investigate colocalization with FcγRIIa and viral particles during ADE

• Functional Validation:

  • Generate TBC1D3G knockout cell lines using CRISPR-Cas9

  • Complement knockout cells with wild-type or mutant TBC1D3G constructs

  • Assess binding of IgG-virus complexes with flow cytometry

  • Measure virus internalization and replication in cells with varying TBC1D3G expression

• Translational Application:

  • Screen inhibitors that disrupt TBC1D3G function to identify potential therapeutic agents against ADE

  • Analyze TBC1D3G expression in patients with severe versus mild dengue infections

What are the technical limitations of current TBC1D3G antibodies?

Current TBC1D3G antibodies have several limitations that researchers should be aware of:

• Specificity challenges: Due to the high sequence similarity between TBC1D3 family members, achieving absolute specificity for TBC1D3G over related proteins (TBC1D3, TBC1D3B, TBC1D3C) remains challenging .

• Validation status: Many commercially available antibodies have limited validation data. Only a few antibodies have been rigorously validated in knockout systems or across multiple applications .

• Application restrictions: Some antibodies are validated only for specific applications such as ELISA or Western blot but not for immunoprecipitation or flow cytometry .

• Species reactivity: Most antibodies primarily target human TBC1D3G with limited cross-reactivity to other species, reflecting its hominoid-specific nature .

• Epitope accessibility: Depending on experimental conditions, certain epitopes may be masked by protein folding or interactions with other molecules.

What emerging methods might improve TBC1D3G research beyond antibody-based approaches?

Several cutting-edge approaches could advance TBC1D3G research beyond traditional antibody methods:

• CRISPR-Cas9 gene editing:

  • Generate endogenously tagged TBC1D3G (with GFP, mCherry, or HaloTag) to track the protein without antibodies

  • Create precise point mutations to study structure-function relationships

• Single-cell transcriptomics:

  • Analyze TBC1D3G expression patterns at single-cell resolution in heterogeneous tissues

  • Functional states of cells expressing TBC1D3G can be explored using databases like CancerSEA

• Proximity-based labeling:

  • BioID or APEX2 fusion proteins to identify the TBC1D3G interactome without relying on antibody-based precipitation

  • Spatial mapping of TBC1D3G interactions in different cellular compartments

• Structural biology approaches:

  • Cryo-EM or X-ray crystallography of TBC1D3G in complex with RAB5 to understand mechanistic details

  • Molecular dynamics simulations to predict functional consequences of mutations

• Multi-omics integration:

  • Combine transcriptomic, proteomic, and metabolomic data to understand TBC1D3G's role in cellular pathways

  • Orthogonal transcriptomics/metabolomics analysis, similar to what revealed TBC1D3G's correlation with elevated glycolytic metabolism in breast cancer cell lines

These emerging methods could provide more comprehensive insights into TBC1D3G's biology while addressing the limitations of current antibody-based approaches.