TBC1D23 Antibody

What is the primary biological function of TBC1D23 in cellular trafficking?

TBC1D23 functions as a critical bridging protein in the endosome-to-Golgi trafficking pathway. It simultaneously binds to golgins (including GOLGA1 and GOLGA4) located at the trans-Golgi and to the WASH complex on endosome-derived vesicles. This dual-binding capability enables TBC1D23 to serve as an adaptor that links vesicle to target membrane, acting as a specificity determinant during endosome-to-Golgi trafficking. The protein facilitates the golgin-mediated capture of vesicles generated using AP-1, demonstrating its essential role in membrane traffic specificity .

How does TBC1D23 differ structurally and functionally from other TBC domain proteins?

Despite containing a TBC domain typically associated with Rab GTPase-activating proteins (GAPs), TBC1D23 is distinctive because it lacks the conserved arginine and glutamine residues essential for Rab GAP activity. This makes TBC1D23 an apparently catalytically inactive member of the TBC family. Additionally, TBC1D23 contains a rhodanese-like domain, which is found in combination with other domains in diverse proteins including phosphatases, heat shock proteins, and ubiquitin hydrolases. Functionally, while other TBC proteins primarily regulate Rab GTPase activity, TBC1D23 serves as a vesicle-golgin adaptor specifically required for endosome-to-Golgi traffic .

What experimental models are most suitable for studying TBC1D23 function?

Based on published research, several experimental models have proven effective for studying TBC1D23:

• Cell lines: HEK-293T, HeLa, HepG2, and U2OS cells all express TBC1D23 and are suitable for in vitro studies .

• CRISPR/Cas9-generated null mutants: Creating TBC1D23 knockout cell lines using CRISPR/Cas9 technology allows for functional studies examining the effects of TBC1D23 deletion on trafficking pathways .

• Mitochondrial relocation assays: Systems where golgins are relocated to mitochondria have been particularly valuable for studying vesicle tethering, as they create an ectopic situation where vesicles accumulate in a tethered state, allowing for detailed analysis of the tethering process .

• Cargo trafficking assays: TGN46 and CI-MPR provide ideal cargo proteins for analyzing endosome-to-Golgi trafficking, as their retrieval can be quantified by both immunofluorescence and immunoblotting .

What are the optimal conditions for immunoprecipitation of TBC1D23?

For effective immunoprecipitation of TBC1D23, researchers should follow these methodological steps:

• Cell preparation: Use cells at approximately 75% confluency (such as 293T cells in two 175 cm² flasks) transfected with plasmids encoding GFP-tagged TBC1D23.

• Lysis buffer composition: Employ a buffer containing 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5 mM EDTA, 0.5% (v/v) Triton X-100, 1 mM PMSF, and complete protease inhibitors.

• Lysis procedure: Harvest cells by centrifugation, wash once in ice-cold PBS, and lyse for 30 minutes at 4°C.

• Clarification: Clarify lysates by centrifugation before proceeding to the immunoprecipitation step.

• Antibody-bead interaction: For GFP-tagged TBC1D23, use 25 μL of packed GFP-Trap beads and incubate for two hours with rotation at 4°C.

• Washing and elution: Wash beads in lysis buffer and elute bound proteins by adding 100 μL of 2× SDS sample buffer.

• Analysis: Analyze eluates by mass spectrometry and immunoblotting to confirm successful immunoprecipitation .

What dilution ranges and applications are recommended for TBC1D23 antibodies in different experimental contexts?

Based on validated antibody protocols, the following application-specific dilutions are recommended:

It is essential to titrate these antibodies in each testing system to obtain optimal results. Cell types with confirmed positive signals include HepG2, HEK-293, HeLa, U2OS, LNCaP, Jurkat, K-562, HSC-T6, and NIH/3T3 cells .

How should TBC1D23 antibodies be stored to maintain optimal reactivity?

For maximum antibody stability and performance:

• Storage temperature: Store at -20°C. Antibodies are stable for one year after shipment at this temperature.

• Storage buffer: Most commercial TBC1D23 antibodies are provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3.

• Aliquoting considerations: For -20°C storage, aliquoting is generally unnecessary, though this may vary by manufacturer.

• Special formulations: Some smaller volume preparations (20 μL) may contain 0.1% BSA for added stability.

• Handling: Minimize freeze-thaw cycles by working with small aliquots when possible.

These storage conditions ensure maintenance of antibody reactivity and specificity for experimental applications .

How can researchers distinguish between multiple endosome-to-Golgi trafficking pathways when studying TBC1D23 function?

Distinguishing between multiple endosome-to-Golgi trafficking pathways requires careful experimental design:

• Parallel pathway analysis: Research indicates that multiple partially redundant mechanisms operate in parallel in endosome-to-Golgi trafficking. For example, GCC88 captures CI-MPR-containing vesicles independently of TBC1D23, suggesting either two types of endosome-to-Golgi carriers with overlapping cargos or two mechanisms for capturing the same vesicles.

• Cargo-specific markers: Utilize different cargo proteins known to traffic through specific pathways. TGN46 and CI-MPR are well-established markers for endosome-to-Golgi trafficking that can be affected by TBC1D23 deletion.

• Component-specific perturbations: Compare the effects of depleting different trafficking components. For instance, both AP-1 clathrin adaptor and sorting nexins SNX1/2 have been reported to direct retrieval of proteins from endosomes to the Golgi.

• Rescue experiments: Perform rescue experiments with truncated forms of TBC1D23 to identify which domains are essential for specific trafficking pathways. For example, a truncated form lacking residues 559-684 that bind to FAM21A cannot restore endosome-to-Golgi traffic in TBC1D23-deficient cells.

• Combined golgin deletions: Delete multiple golgins simultaneously (e.g., golgin-97 and golgin-245) to observe the combined effect on trafficking pathways, as individual deletions may show redundancy .

What are the methodological approaches to investigate the role of TBC1D23 in brain development and neurite outgrowth?

To investigate TBC1D23's role in neurodevelopment, researchers should consider these methodological approaches:

• Human genetic studies: Analyze patients with truncating mutations in TBC1D23, which have been linked to impaired brain development but are not cell-lethal, suggesting additional mechanisms exist to sustain endosome-to-Golgi traffic.

• Animal models: Develop mouse models with TBC1D23 mutations to study effects on cortical neuron positioning and neurite outgrowth.

• Primary neuronal cultures: Establish primary neuronal cultures from wild-type and TBC1D23-deficient models to directly assess neurite outgrowth and morphology.

• Vesicular trafficking assays in neurons: Adapt vesicular trafficking assays to neuronal systems to understand how TBC1D23 contributes to membrane trafficking and cargo delivery, processes essential for axonal and dendritic growth.

• Imaging techniques: Employ advanced imaging techniques such as live-cell microscopy to visualize trafficking events in developing neurons.

• Molecular interaction studies: Investigate TBC1D23 interactions with neuronal-specific partners that might regulate its function in the developing brain .

How does the interaction between TBC1D23 and the WASH complex specifically contribute to endosomal trafficking?

The interaction between TBC1D23 and the WASH complex represents a critical mechanism in endosomal trafficking that can be studied through these methodological approaches:

• Proximity biotinylation: Apply BirA* fusion to the N- or C-terminus of TBC1D23 to identify interacting proteins. This approach has revealed that TBC1D23 specifically biotinylates golgin-97, golgin-245, FAM91A1, and the cargo of endosome-derived carriers.

• Domain mapping: The C-terminus of TBC1D23 (specifically residues 559-684) binds to FAM21A, a component of the WASH complex. Truncated forms of TBC1D23 lacking these residues fail to restore endosome-to-Golgi traffic in TBC1D23-deficient cells.

• Mitochondrial relocation assays: Relocate TBC1D23 to mitochondria (TBC1D23-mito) to visualize its ability to recruit endosome-derived carriers and the WASH complex. Electron microscopy reveals an accumulation of circular and oval membranes tethered between the mitochondria.

• WASH complex depletion studies: Deplete FAM21A to confirm that the WASH complex is required for normal endosome-to-Golgi traffic of CI-MPR and TGN46.

• Vesicle capture analysis: In TBC1D23-deficient cells, mitochondrial capture of endosomal vesicles (visualized by the endosome-to-Golgi cargo TGN46) is markedly reduced but can be rescued by reintroduction of TBC1D23 .

What are the common pitfalls in TBC1D23 immunodetection and how can researchers overcome them?

Several common challenges arise in TBC1D23 immunodetection experiments:

• Antibody specificity concerns: Validate antibody specificity using positive and negative controls. CRISPR/Cas9-generated TBC1D23 null mutants serve as excellent negative controls to confirm antibody specificity.

• Variable expression levels: TBC1D23 expression levels may vary across cell types and tissues. Use appropriate positive controls (such as HeLa, HepG2, or HEK-293 cells) that reliably express detectable levels of TBC1D23.

• Signal strength issues in immunohistochemistry: For IHC applications, antigen retrieval is critical. Use TE buffer pH 9.0 or alternatively citrate buffer pH 6.0 as recommended for optimal results.

• Background in immunofluorescence: For cleaner IF/ICC signals, optimize fixation methods (typically paraformaldehyde fixation), permeabilization conditions, and blocking buffer composition. Antibody dilutions between 1:50-1:500 have been validated for IF applications.

• Molecular weight variability: TBC1D23 may appear between 70-78 kDa on Western blots due to post-translational modifications or splice variants. The calculated molecular weight is 78 kDa (699 amino acids) .

How can researchers validate functional assays for TBC1D23 in vesicular trafficking studies?

To ensure reliable functional assays for TBC1D23 in vesicular trafficking:

What is the relationship between TBC1D23 mutations and pontocerebellar hypoplasia, and how can this be studied?

The relationship between TBC1D23 mutations and pontocerebellar hypoplasia requires multifaceted research approaches:

• Genetic analysis: Perform comprehensive genetic sequencing in patients with pontocerebellar hypoplasia to identify homozygous mutations in TBC1D23. Studies have shown that biallelic mutations in TBC1D23 lead to a non-degenerative form of pontocerebellar hypoplasia.

• Neuroimaging correlation: Correlate specific TBC1D23 mutations with neuroimaging findings, including severe volume loss of pons and cerebellum, hypoplasia of cortex, and corpus callosum abnormalities.

• Clinical phenotyping: Document the constellation of neurological manifestations, including psychomotor impairment, microcephaly, brainstem deficits, and ataxia. Also note systemic findings like recurrent respiratory infections, which are common in affected individuals.

• Functional validation: Test the trafficking defects associated with patient-derived TBC1D23 mutations in cellular models to establish causality.

• Animal models: Develop animal models harboring TBC1D23 mutations to study developmental consequences on brain architecture and function.

• Therapeutic explorations: Investigate whether enhancing alternative trafficking pathways might compensate for TBC1D23 deficiency .

How might TBC1D23's role in innate immunity signaling impact experimental design in immunological research?

TBC1D23's reported inhibitory effects on innate immunity require specific experimental considerations:

• Baseline vs. stimulated conditions: Compare inflammatory markers and cytokine levels in TBC1D23-deficient subjects at baseline and following stimulation, as baseline differences may not be significant.

• Time-course analyses: TBC1D23 does not alter initial activation events but affects maintenance of inflammatory gene expression several hours after bacterial lipopolysaccharide (LPS) challenge. Design experiments with appropriate time points (extending several hours post-stimulation).

• Multiple TLR pathway assessment: Evaluate multiple TLR and dectin/CLEC7A-signaling pathways, as TBC1D23 has been reported to inhibit multiple such pathways.

• Infection models: Assess whether TBC1D23 deficiency exacerbates inflammatory responses to bacterial infections, potentially explaining the clinical finding of recurrent respiratory infections in patients with TBC1D23 mutations.

• Cytokine profiling: Perform comprehensive cytokine profiling rather than focusing on individual inflammatory mediators.

• Mechanistic studies: Investigate how TBC1D23's vesicular trafficking function might intersect with innate immune signaling pathways .