TBC1D31 Antibody

What is TBC1D31 and why is it important in research?

TBC1D31 (TBC1 Domain Family, Member 31) is a protein that belongs to the Tre2/Bub2/Cdc16 (TBC) domain-containing family. It functions as a Rab GTPase activating protein (GAP) involved in vesicle-mediated transport processes. Research has identified TBC1D31 as a critical regulator of endolysosomal trafficking, particularly in the context of EGFR (Epidermal Growth Factor Receptor) degradation pathways . The importance of studying TBC1D31 has increased significantly since genomic amplification at the 8q24.13 locus (where TBC1D31 is located) was found to promote hepatocellular carcinoma (HCC) development, making it a potential oncogene and therapeutic target .

What types of TBC1D31 antibodies are available for research?

Several types of TBC1D31 antibodies are available for research purposes, including polyclonal antibodies targeting different amino acid regions of the protein. These include antibodies recognizing amino acids 662-1001, 330-678, and 1-340 regions . These antibodies come in various conjugated forms, including unconjugated, biotin-conjugated, FITC-conjugated, and HRP-conjugated versions, offering flexibility for different experimental applications . The availability of antibodies targeting different epitopes enables researchers to investigate the localization, expression levels, and interactions of TBC1D31 through various techniques.

Which applications are TBC1D31 antibodies commonly used for?

TBC1D31 antibodies are typically employed in several research applications, including Enzyme-Linked Immunosorbent Assay (ELISA), Immunohistochemistry (IHC), and Western Blotting (WB) . ELISA applications allow for quantitative assessment of TBC1D31 levels in biological samples. IHC enables visualization of TBC1D31 expression patterns in tissue sections, which has been valuable in comparing expression levels between tumor and non-tumor tissues . Western blotting facilitates detection of TBC1D31 protein levels and post-translational modifications in cell and tissue lysates. The choice of application depends on the specific research question being addressed.

How can TBC1D31 antibodies be used to investigate its role in vesicular trafficking?

To investigate TBC1D31's role in vesicular trafficking, researchers can employ a multi-faceted approach combining immunofluorescence and co-localization studies. TBC1D31 antibodies can be used alongside markers for different endosomal compartments (such as EEA1 for early endosomes) to track the progression of cargo (like EGFR) through the endocytic pathway . Time-course experiments following EGF stimulation can reveal how TBC1D31 affects EGFR trafficking dynamics. Researchers should establish baseline trafficking patterns in control cells, then compare with TBC1D31-depleted or overexpressing cells to identify specific steps disrupted by TBC1D31 manipulation. Complementary approaches include electron microscopy with immunogold labeling using TBC1D31 antibodies to visualize its subcellular localization at ultrastructural resolution.

What methodologies can be employed to study the interaction between TBC1D31 and Rab GTPases?

To study interactions between TBC1D31 and Rab GTPases (particularly Rab22A), researchers can implement several complementary approaches. Co-immunoprecipitation assays using TBC1D31 antibodies can pull down protein complexes for subsequent identification of associated Rab proteins. In vitro GAP assays can directly measure TBC1D31's ability to catalyze GTP hydrolysis for specific Rab proteins, measuring the conversion of GTP to GDP . Proximity ligation assays (PLA) offer another powerful approach to visualize TBC1D31-Rab interactions in situ with subcellular resolution. Researchers should also consider using FRET (Förster Resonance Energy Transfer) or BRET (Bioluminescence Resonance Energy Transfer) to detect direct interactions in living cells, providing insights into the dynamics of these interactions.

How can TBC1D31 antibodies be utilized to investigate its role in cancer pathogenesis?

Investigating TBC1D31's role in cancer pathogenesis requires a comprehensive approach. Immunohistochemistry using TBC1D31 antibodies on tissue microarrays containing tumor and adjacent normal tissues can establish correlation between TBC1D31 expression and clinical parameters like tumor stage, grade, and patient survival . For mechanistic studies, researchers should combine immunoblotting analysis of TBC1D31 with downstream signaling markers (p-EGFR, p-AKT, p-ERK1/2) following manipulation of TBC1D31 levels in cancer cell lines . Xenograft models with TBC1D31-manipulated cancer cells can help assess its impact on tumor growth and metastasis in vivo. Researchers should also explore the relationship between TBC1D31 expression and drug resistance by comparing antibody-detected protein levels with response to targeted therapies like EGFR inhibitors or lenvatinib .

What controls should be included when using TBC1D31 antibodies in experiments?

When designing experiments with TBC1D31 antibodies, incorporating proper controls is essential. For immunoblotting, include positive controls (cell lines known to express TBC1D31, such as HCC cell lines), negative controls (cell lines with TBC1D31 knockdown), and loading controls (housekeeping proteins like β-actin or GAPDH) . For immunohistochemistry, include isotype controls (using non-specific IgG of the same species and concentration) to control for non-specific binding. Include antigen competition controls where the antibody is pre-incubated with its immunizing peptide to demonstrate binding specificity . For functional studies, compare multiple TBC1D31 antibodies targeting different epitopes to ensure consistent results. Additionally, validate antibody specificity by confirming reduced signal in TBC1D31 knockdown samples compared to controls.

What is the optimal protocol for immunoprecipitation using TBC1D31 antibodies?

For optimal immunoprecipitation (IP) of TBC1D31, begin with cell lysis using a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, and protease/phosphatase inhibitors. Pre-clear the lysate with Protein G beads for 1 hour at 4°C to reduce non-specific binding. Incubate 1-2 mg of pre-cleared lysate with 2-5 μg of TBC1D31 antibody overnight at 4°C with gentle rotation. Since the antibody is Protein G purified, add 30-50 μl of Protein G magnetic beads and incubate for 2-3 hours at 4°C . Wash the beads 4-5 times with ice-cold lysis buffer, then elute bound proteins by boiling in 2X Laemmli sample buffer. For co-IP experiments investigating TBC1D31-Rab22A interactions, modify the lysis buffer to include 5 mM MgCl₂ to preserve GTPase activity. Validate IP efficiency by immunoblotting a small portion of the eluate with a second TBC1D31 antibody targeting a different epitope.

How should researchers optimize immunohistochemistry protocols for TBC1D31 detection in tissue samples?

To optimize immunohistochemistry protocols for TBC1D31 detection, begin with antigen retrieval in citrate buffer (pH 6.0) using a pressure cooker for 15-20 minutes. Block endogenous peroxidase activity with 3% hydrogen peroxide and prevent non-specific binding with 5% normal serum from the same species as the secondary antibody. Incubate sections with primary TBC1D31 antibody at optimized dilutions (typically starting at 1:100-1:500) overnight at 4°C . Apply biotinylated secondary antibody if using unconjugated primary antibody, or proceed directly with chromogenic detection if using conjugated antibodies (HRP or biotin-conjugated) . For biotin-conjugated TBC1D31 antibodies, apply streptavidin-HRP complex. Optimize antibody dilution, incubation time, and antigen retrieval conditions for each tissue type. Include positive control tissues (like HCC samples known to overexpress TBC1D31) and validate staining specificity by correlating IHC results with parallel Western blot analyses .

What are common challenges when working with TBC1D31 antibodies and how can they be addressed?

Several challenges can arise when working with TBC1D31 antibodies. High background in immunostaining can be addressed by increasing blocking time (2-3 hours), using alternative blocking agents (5% BSA or 10% normal serum), or implementing additional washing steps. For weak signals, optimize antibody concentration through titration experiments, extend primary antibody incubation time, or enhance detection using amplification systems like tyramide signal amplification. Cross-reactivity issues can be overcome by pre-adsorbing the antibody with potential cross-reactive proteins or using antibodies targeting unique epitopes of TBC1D31 . Poor reproducibility between experiments often stems from variations in sample preparation; standardize lysate preparation methods and protein quantification techniques. For unexpected molecular weight bands, verify if they represent post-translationally modified forms, degradation products, or splice variants of TBC1D31 by comparing with positive controls and literature references.

How should researchers interpret discrepancies between TBC1D31 mRNA and protein expression data?

When interpreting discrepancies between TBC1D31 mRNA and protein expression data, researchers should consider several factors. Post-transcriptional regulation mechanisms (miRNAs, RNA-binding proteins) may affect translation efficiency. Post-translational modifications and protein stability can significantly influence protein levels without affecting mRNA abundance. In HCC research specifically, evaluate if genomic amplification at 8q24.13 corresponds with both increased mRNA and protein expression of TBC1D31, as observed in previous studies . Calculate correlation coefficients between mRNA and protein levels across multiple samples to quantify the degree of discrepancy. Consider temporal dynamics, as mRNA and protein turnover rates differ substantially. Analyze samples at multiple time points following experimental manipulation to capture these differences. Validate findings using multiple methodologies (RT-qPCR, RNA-seq for mRNA; Western blot, mass spectrometry for protein) to confirm discrepancies are biological rather than technical artifacts.

What approaches can be used to validate TBC1D31 antibody specificity?

To validate TBC1D31 antibody specificity, implement a multi-faceted approach. Perform Western blot analysis on lysates from cells with TBC1D31 knockdown (using siRNA or CRISPR-Cas9) compared to control cells; a specific antibody will show significantly reduced signal in knockdown samples . Conduct peptide competition assays by pre-incubating the antibody with excess immunizing peptide (662-1001AA region for ABIN7371151) before application; specific binding will be blocked . Compare staining patterns using multiple antibodies targeting different TBC1D31 epitopes; consistent patterns suggest specificity. Verify that immunoprecipitation with the antibody pulls down a protein of the expected molecular weight that can be detected by a different TBC1D31 antibody. For mass spectrometry validation, immunoprecipitate TBC1D31 and confirm its identity in the pulled-down material. Consider heterologous expression systems by transfecting TBC1D31-negative cell lines with tagged TBC1D31 constructs and confirming antibody recognition.

What is known about TBC1D31's role in EGFR trafficking and how can researchers further investigate this pathway?

Recent research has established TBC1D31 as a critical regulator of EGFR trafficking through its GAP activity on Rab22A. TBC1D31 hydrolyzes Rab22A-bound GTP, thereby reducing Rab22A-mediated trafficking of EGFR from early endosomes to late endosomes/lysosomes for degradation . This mechanism leads to delayed EGFR degradation, prolonged EGFR signaling, and enhanced downstream pathway activation (particularly AKT and ERK1/2) . To further investigate this pathway, researchers should employ live-cell imaging with fluorescently-tagged EGFR and endosomal markers in cells with manipulated TBC1D31 levels. Pulse-chase experiments using biotinylated EGF or antibody-based approaches can quantify EGFR internalization and degradation rates. Structure-function analyses using TBC1D31 mutants (particularly in the TBC domain) can identify critical residues required for Rab22A interaction. Proximity labeling techniques (BioID or APEX2) coupled with mass spectrometry can map the TBC1D31 interactome at endosomal compartments, potentially revealing additional components of this regulatory pathway.

How does TBC1D31 contribute to cancer development and potential therapeutic strategies?

TBC1D31 contributes to cancer development through genomic amplification at 8q24.13, which drives its overexpression in hepatocellular carcinoma (HCC) . Functionally, TBC1D31 promotes tumor growth and metastasis by enhancing EGFR signaling persistence through reduced lysosomal degradation of the receptor . This leads to sustained activation of downstream oncogenic pathways, including AKT and ERK1/2 signaling . Additionally, TBC1D31 overexpression increases resistance to therapeutics like lenvatinib in HCC cells . Potential therapeutic strategies include developing small molecule inhibitors targeting TBC1D31's GAP activity, employing RNA interference to reduce TBC1D31 expression, or combining EGFR-targeted therapies with TBC1D31 inhibition to overcome resistance. In experimental models, TBC1D31 inhibition restores sensitivity to lenvatinib, suggesting combination therapy approaches may be effective in patients with TBC1D31 amplification or overexpression . Researchers should explore these strategies using patient-derived xenograft models and correlate TBC1D31 expression with treatment response in clinical samples.

What are the emerging techniques for studying TBC1D31 localization and dynamics in living cells?

Emerging techniques for studying TBC1D31 localization and dynamics in living cells offer unprecedented insights into its function. CRISPR-Cas9 genome editing to add fluorescent protein tags at the endogenous TBC1D31 locus allows visualization of physiologically relevant expression and localization patterns. Super-resolution microscopy techniques (STED, PALM, STORM) can resolve TBC1D31's subcellular distribution at nanometer resolution, particularly in relation to endosomal compartments. Optogenetic approaches enable acute, reversible activation or inhibition of TBC1D31 function in specific subcellular locations. Fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) can measure TBC1D31's diffusion dynamics and binding kinetics to membranes or protein partners. For studying TBC1D31's impact on vesicular trafficking dynamics, lattice light-sheet microscopy offers superior temporal resolution with reduced phototoxicity. Single-particle tracking of fluorescently labeled endosomes in TBC1D31-manipulated cells can reveal changes in trafficking routes and kinetics. These approaches will provide crucial insights into TBC1D31's dynamic regulation of vesicular trafficking pathways.

How does TBC1D31 antibody selection impact experimental outcomes across different model systems?

The selection of TBC1D31 antibodies significantly impacts experimental outcomes across different model systems. Antibodies targeting different epitopes (e.g., AA 662-1001 versus AA 330-678) may yield varying results due to epitope accessibility differences in fixed versus live tissues, or between species . Polyclonal antibodies offer broader epitope recognition but potential batch-to-batch variability, while monoclonal antibodies provide consistency but might miss certain protein conformations. When transitioning between in vitro cell culture and in vivo tissue analysis, optimize antibody concentration and detection methods for each system. For cross-species studies, verify epitope conservation through sequence alignment analysis between human and model organism TBC1D31. The table below summarizes recommended antibody applications across experimental systems:

What strategies should be employed when comparing TBC1D31 expression across different cancer types?

When comparing TBC1D31 expression across different cancer types, researchers should implement a multi-platform approach. Begin with in silico analysis of publicly available transcriptomic datasets (TCGA, GEO) to identify cancer types with TBC1D31 genomic amplification or mRNA overexpression. Follow with tissue microarray analysis using validated TBC1D31 antibodies, applying standardized staining and scoring protocols across all cancer types . Quantify staining intensity and percentage of positive cells using digital pathology tools to minimize subjective assessment. Control for tissue-specific factors by including matched normal tissues for each cancer type and normalize expression accordingly. Correlate TBC1D31 protein levels with copy number data to identify amplification-driven overexpression. For mechanistic insights, compare EGFR expression and downstream signaling markers across cancer types to determine if the TBC1D31-EGFR axis is consistently operational . Validate key findings in representative cell lines from each cancer type using knockdown experiments and functional assays. This systematic approach will reveal whether TBC1D31's oncogenic role extends beyond hepatocellular carcinoma to other malignancies.

How might TBC1D31 antibodies be utilized in developing targeted cancer therapies?

TBC1D31 antibodies could play crucial roles in developing targeted cancer therapies through several avenues. As diagnostic tools, they can identify patients with TBC1D31 overexpression who might benefit from targeted approaches, particularly those with resistance to existing therapies like lenvatinib . For therapeutic development, TBC1D31 antibodies can facilitate high-throughput screening assays to identify small molecule inhibitors of TBC1D31's GAP activity. Antibody-drug conjugates targeting cell-surface proteins regulated by TBC1D31 could provide selective delivery of cytotoxic payloads to cancer cells. In combination therapy research, TBC1D31 antibodies can monitor protein levels and pathway activation to optimize timing and dosing of EGFR inhibitors with TBC1D31-targeting agents . For resistance mechanisms, serial biopsies analyzed with TBC1D31 antibodies can track expression changes during treatment and disease progression. Additionally, these antibodies can validate in vivo efficacy of TBC1D31-targeted therapies in preclinical models by confirming target engagement and downstream pathway modulation.