TRAPPC8 Antibody

What is TRAPPC8 and what is its biological significance?

TRAPPC8 (Trafficking Protein Particle Complex 8) functions as a critical component of the TRAPPIII complex involved in membrane trafficking pathways. This large protein (approximately 150 kDa) plays essential roles in vesicle-mediated transport and has been identified as a host factor required for human papillomavirus (HPV) infection . Research has demonstrated that TRAPPC8 participates in the early stages of HPV infection, including viral entry and intracellular trafficking . The protein exhibits dual localization patterns - partially on the cell surface where it can interact with viral particles, and within intracellular compartments including the Golgi apparatus .

Protein identification details include:

• UniProt ID: Q9Y2L5 (Human)

• Entrez Gene ID: 22878 (Human)

• Aliases: GSG1, HsT2706, KIAA1012, TRS85

What are the validated applications for TRAPPC8 antibodies?

Based on comprehensive validation studies, TRAPPC8 antibodies have demonstrated effectiveness in multiple experimental applications:

Selection should be based on specific experimental needs and validation data available for each antibody.

How can researchers validate TRAPPC8 antibody specificity?

Robust validation of TRAPPC8 antibody specificity requires multiple complementary approaches:

• siRNA knockdown verification: Transfect cells with TRAPPC8-specific siRNAs (such as siGENOME set) and confirm reduction of the detected protein band by Western blot .

• Multiple antibody approach: Utilize antibodies targeting different TRAPPC8 epitopes (N-terminal, central, and C-terminal regions) to confirm consistent detection patterns .

• Immunogen sequence verification: Confirm antibody recognition of the specific immunogen sequence. For example, PA5-59429 targets the sequence: "DYDLNISATTPWFESYRETFLQSMPASDHEF LNHYLACMLVASSSEAEPVEQFSKLSQEQHRIQHNSDYSYPKWFIPNTLKYYVLLHDVSAGDE" .

• Cross-species reactivity assessment: Verify expected cross-reactivity with orthologs. The PA5-59429 antibody shows 95% sequence identity with mouse and 96% with rat orthologs .

• Control experiments: Always include appropriate positive and negative controls in all experimental designs.

What methodological approaches are optimal for detecting TRAPPC8 in subcellular localization studies?

For precise subcellular localization of TRAPPC8:

• Surface vs. internal pool differentiation:

  • For surface TRAPPC8: Use non-permeabilized cells with antibodies targeting exposed epitopes (anti-P880/894 has been validated for cell surface detection) .

  • For total TRAPPC8: Use permeabilized cells to detect both internal and surface pools .

• Confocal microscopy optimization:

  • Utilize Z-stack analysis to distinguish between surface and intracellular localization.

  • For co-localization with viral particles or cellular organelles, employ dual immunofluorescence techniques.

  • The central region antibody (anti-P880/894) is particularly effective for detecting surface TRAPPC8, while anti-N1/603 works better for intracellular detection .

• Experimental design considerations:

  • For viral interaction studies, synchronize viral attachment by performing infections at 4°C for 1 hour before shifting to 37°C .

  • Include TRAPPC8 knockdown cells as specificity controls .

  • For Golgi localization studies, use appropriate markers for different Golgi compartments.

Flow cytometry data has confirmed that the epitope region of anti-P880/894 (aa 880–894) is exposed on the cell surface, while other regions may be less accessible .

How should researchers optimize TRAPPC8 antibodies for protein interaction studies?

For effective protein interaction studies involving TRAPPC8:

• Co-immunoprecipitation protocol optimization:

  • Use lysis buffers containing mild detergents (1% NP-40) to preserve protein-protein interactions.

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding.

  • For viral protein interactions (such as HPV L2), consider using epitope-tagged constructs for easier pulldown .

• Controls and validation:

  • Always include IgG control, input samples, and TRAPPC8 knockdown cells as controls.

  • Confirm interactions using reciprocal co-immunoprecipitation approaches.

  • Validate interaction specificity through mutagenesis of key binding regions.

• Interaction partner identification:

  • TRAPPC8 has been demonstrated to interact with HPV L2 proteins, with clear specificity for certain variants (MaL2 but not NuL2) .

  • TRAPPC8 co-precipitates with other TRAPP complex components like TRAPPC12, indicating it functions as part of a larger complex .

• Methodological improvements:

  • Consider crosslinking approaches for capturing transient interactions.

  • For temporal analysis of interactions, perform time-course experiments following stimulation or infection.

What are the critical considerations for quantitative Western blot analysis of TRAPPC8?

For accurate quantitative Western blotting of TRAPPC8:

• Technical optimizations for large proteins:

  • Use low percentage gels (6-8%) to properly resolve the ~150 kDa TRAPPC8 protein.

  • Extend transfer time or use specialized transfer systems for efficient transfer of large proteins.

  • Validate molecular weight using knockdown controls to confirm specificity .

• Antibody selection and optimization:

  • Determine optimal antibody dilutions empirically for each application.

  • The following antibodies have been validated for Western blot: anti-N1/603, anti-P880/894, and anti-P1270/1285 .

  • Include appropriate loading controls (α-tubulin has been validated) .

• Quantification considerations:

  • Establish the linear detection range using serial dilutions of cell lysates.

  • For comparison between samples, normalize to multiple housekeeping proteins.

  • Consider potential post-translational modifications that might affect protein migration or antibody recognition.

• Experimental design:

  • For knockdown validation experiments, optimal results are typically observed 48-72 hours post-transfection with TRAPPC8 siRNAs .

How can TRAPPC8 antibodies be utilized to investigate HPV infection mechanisms?

TRAPPC8 antibodies have proven valuable for investigating HPV infection mechanisms:

• Studying viral entry pathways:

  • TRAPPC8 knockdown experiments have demonstrated its requirement for HPV entry .

  • Surface TRAPPC8 colocalizes with HPV pseudovirions (PsVs) during early viral attachment, detectable with anti-P880/894 antibody .

  • Entry assays combining TRAPPC8 antibodies with viral capsid detection provide insights into internalization mechanisms.

• Quantifying infection efficiency:

  • TRAPPC8 knockdown reduces transduction efficiency of HPV pseudovirions and authentic HPV31 virion infection .

  • Combined immunofluorescence and flow cytometry approaches can assess the correlation between TRAPPC8 levels and infection rates.

• Tracking intracellular viral trafficking:

  • Co-immunoprecipitation studies have established that TRAPPC8 interacts with HPV L2 capsid protein .

  • This interaction appears to be a general property of L2 proteins from multiple HPV types (HPV16, HPV31, HPV51) .

  • Time-course experiments using TRAPPC8 antibodies can track the dynamics of viral trafficking through cellular compartments.

• Experimental models:

  • Both transformed cell lines (HeLa) and keratinocytes (HaCaT) show TRAPPC8-dependent HPV infection .

  • Authentic virion experiments confirm findings from pseudovirion models .

What role does TRAPPC8 play in Golgi dynamics during viral infection?

TRAPPC8's involvement in Golgi dynamics during viral infection can be investigated using specialized approaches:

• Visualizing Golgi morphology changes:

  • Expression of GFP-fused L2 (which interacts with TRAPPC8) induces dispersal of Golgi stack structure in HeLa cells .

  • This phenotype is similar to that observed with TRAPPC8 knockdown, suggesting L2 binding inhibits TRAPPC8 function .

• Investigating escape mechanisms:

  • Research suggests that L2-TRAPPC8 interaction may cause Golgi destabilization, potentially assisting HPV genome escape from the trans-Golgi network .

  • Immunofluorescence studies with Golgi markers can track these changes temporally.

• TRAPPIII complex analysis:

  • TRAPPC8 functions as part of the TRAPPIII complex, with interactions with other subunits like TRAPPC12 .

  • Combined knockdown studies of TRAPPC8 and TRAPPC12 demonstrate similar effects on viral infection, suggesting the importance of the entire complex .

• Methodological approach:

  • Use antibodies against both TRAPPC8 and Golgi markers (such as GM130 for cis-Golgi or TGN46 for trans-Golgi).

  • Time-course experiments following viral infection can track progressive changes in Golgi architecture.

How should researchers address inconsistent results with different TRAPPC8 antibodies?

When encountering inconsistent results with different TRAPPC8 antibodies:

• Epitope accessibility analysis:

  • Different antibodies target distinct regions of TRAPPC8 with varying accessibility :

    • Anti-N1/603: N-terminal region (aa 1-603)

    • Anti-P880/894: Central region (aa 880-894)

    • Anti-P1270/1285: C-terminal region (aa 1270-1285)

  • Surface detection works best with anti-P880/894, while intracellular detection may require other antibodies .

• Application-specific optimization:

  • For Western blotting: Anti-N1/603 shows strong detection of the 150 kDa band.

  • For immunofluorescence: Anti-P880/894 is effective for surface detection, while anti-N1/603 works better for permeabilized cells .

  • For co-immunoprecipitation: The choice depends on the interaction being studied.

• Experimental conditions:

  • Fixation methods significantly impact epitope accessibility (PFA vs. methanol).

  • Blocking reagents and antibody dilutions should be optimized for each antibody.

  • Cell types may exhibit different TRAPPC8 expression patterns or post-translational modifications.

• Validation approaches:

  • Always include TRAPPC8 knockdown controls.

  • Consider commercial antibodies with validated applications (PA5-59429, HPA041107, NBP1-93708) .

What methodological considerations are important for TRAPPC8 knockdown experiments?

For effective TRAPPC8 knockdown experiments:

• siRNA selection and optimization:

  • Multiple validated siRNAs are available (KIAA1012-01, -02, -03, or -04) .

  • Optimal knockdown is typically achieved 48-72 hours post-transfection .

  • Transfection efficiency varies by cell type (HeLa vs. HaCaT cells require different protocols) .

• Knockdown verification:

  • Verify TRAPPC8 reduction by Western blot using anti-N1/603 antibody .

  • Also confirm functional consequences (such as altered Golgi morphology or reduced viral infection).

  • Monitor cell viability, as complete TRAPPC8 depletion may affect cell health in some systems.

• Experimental design:

  • Include appropriate controls (non-targeting siRNA) .

  • For viral infection studies, inoculate cells 48 hours post-transfection for optimal results .

  • For authentic virion experiments, measure E1^E4 transcripts by RT-qPCR to quantify infection .

• Rescue experiments:

  • Consider complementation with siRNA-resistant TRAPPC8 constructs to confirm specificity.

  • Domain mutants can help identify functional regions of the protein.

How can TRAPPC8 antibodies be employed in developing novel antiviral strategies?

The role of TRAPPC8 in HPV infection suggests potential applications in antiviral research:

• Target validation approaches:

  • TRAPPC8 antibodies can validate this protein as a therapeutic target through neutralization experiments.

  • Epitope mapping using domain-specific antibodies can identify critical interaction regions.

• High-throughput screening:

  • Develop cell-based assays using TRAPPC8 antibodies to screen for compounds that disrupt viral-host interactions.

  • Immunofluorescence-based screens can identify molecules that alter TRAPPC8 localization or function.

• Mechanistic studies:

  • Investigate whether TRAPPC8 functions are required for other viral infections beyond HPV.

  • The central region (aa 880-894) appears particularly important for viral interactions and could be targeted specifically .

• Experimental design considerations:

  • Compare multiple HPV types (HPV16, HPV31, HPV51) to establish conservation of mechanisms .

  • Utilize both pseudovirus and authentic virion systems to confirm findings .

What are the future directions for TRAPPC8 antibody development and applications?

Emerging areas for TRAPPC8 antibody research include:

• Improved reagent development:

  • Generation of monoclonal antibodies against specific functional domains.

  • Development of conformation-specific antibodies that recognize native TRAPPC8 complexes.

  • Creation of phospho-specific antibodies to investigate regulatory mechanisms.

• Advanced imaging applications:

  • Single-molecule localization microscopy to precisely map TRAPPC8 distribution.

  • Live-cell compatible antibody fragments for dynamic tracking studies.

  • Multi-color super-resolution microscopy for detailed protein complex analysis.

• Translational research:

  • Investigation of TRAPPC8 expression patterns in HPV-associated cancers.

  • Development of diagnostic approaches based on TRAPPC8 detection.

  • Exploration of TRAPPC8's roles beyond viral infection in cellular physiology and disease.

• System biology approaches:

  • Combining TRAPPC8 antibodies with proximity labeling techniques for comprehensive interactome analysis.

  • Proteomic profiling of TRAPPC8 complexes under different cellular conditions.