TRAPPC2L Antibody

What is TRAPPC2L and why is it important in cellular research?

TRAPPC2L (Trafficking Protein Particle Complex Subunit 2-Like Protein) is a highly conserved core subunit of the TRAPP (TRAnsport Protein Particle) complexes II and III that function as critical tethering factors during membrane trafficking . TRAPPC2L was initially identified based on homology to the TRAPPC2 core subunit and appears to function in post-Golgi compartments . Despite sharing structural similarities with TRAPPC2, functional studies indicate TRAPPC2L has distinct roles, making it an important target for understanding cellular trafficking mechanisms . Mutations in TRAPPC2L have been linked to neurodevelopmental disorders, highlighting its clinical relevance in genetic and neurological research .

What are the primary applications for TRAPPC2L antibodies in cellular research?

TRAPPC2L antibodies are valuable tools for multiple research applications including:

• Immunohistochemistry (IHC): Typically used at dilutions of 1:20-1:50 to visualize TRAPPC2L distribution in tissue samples .

• Immunofluorescence (IF): Effective at concentrations of 0.25-2 μg/mL for subcellular localization studies .

• Western Blot (WB): Recommended dilutions of 1:500-1:2000 for protein expression analysis .

• ELISA: Can be used at high dilutions (1:20000-1:80000) for quantitative detection .

These applications enable researchers to investigate TRAPPC2L expression patterns, subcellular localization, protein interactions, and functional roles in normal and pathological conditions .

How do TRAPPC2L and TRAPPC2 differ functionally despite their structural similarities?

Despite sharing sequence homology, TRAPPC2L and TRAPPC2 are functionally distinct proteins. Biochemical and complementation studies demonstrate that:

• TRAPPC2L and TRAPPC2 genes are found in pairs across species and show broad and overlapping expression patterns .

• Yeast complementation studies confirm functional differences between these proteins .

• While knockdown of either protein in HeLa cells leads to Golgi fragmentation (implicating both in Golgi dynamics), gradient fractionation reveals differential subcellular distribution - TRAPPC2L associates with very low-density membrane fractions that do not contain Golgi markers, unlike TRAPPC2 .

• TRAPPC2L may preferentially associate with specific TRAPP complexes to regulate their activity, potentially in post-Golgi compartments .

These distinctions are important when designing experiments targeting specific TRAPP complex functions and interpreting antibody-based detection results.

What optimization steps are recommended when using TRAPPC2L antibodies for immunohistochemistry?

When optimizing TRAPPC2L antibodies for immunohistochemistry, researchers should consider:

• Dilution optimization: Start with the manufacturer's recommended range (1:20-1:50 for polyclonal antibodies) and perform a dilution series to determine optimal signal-to-noise ratio .

• Antigen retrieval methods: Since TRAPPC2L is involved in membrane trafficking, appropriate membrane permeabilization is crucial. Compare heat-induced epitope retrieval in citrate buffer (pH 6.0) versus EDTA buffer (pH 9.0).

• Incubation conditions: Test both overnight incubation at 4°C and shorter incubations (1-2 hours) at room temperature.

• Detection systems: Compare the sensitivity of different detection methods (e.g., HRP-polymer versus biotin-streptavidin systems).

• Controls: Always include positive controls (tissues known to express TRAPPC2L) and negative controls (antibody diluent only or isotype control) .

Antibody validation using tissues with varying TRAPPC2L expression levels is critical as the Human Protein Atlas project has extensively characterized these antibodies across hundreds of normal and disease tissues .

How can researchers effectively troubleshoot non-specific binding when using TRAPPC2L antibodies?

Non-specific binding can significantly impact the interpretation of TRAPPC2L detection. To address this issue:

• Block adequately: Optimize blocking conditions using 5-10% normal serum from the same species as the secondary antibody, with optional addition of 0.1-0.3% Triton X-100 for membrane permeabilization.

• Validate antibody specificity: Test the antibody against recombinant TRAPPC2L protein and TRAPPC2 to ensure specificity, as both share structural similarities. Commercial TRAPPC2L antibodies have been tested on protein arrays of 364 human recombinant protein fragments to validate specificity .

• Adjust antibody concentration: Titrate to find the optimal concentration that provides specific signal while minimizing background (typically 1:500-1:2000 for Western blotting) .

• Increase washing steps: Implement additional and longer wash steps with 0.1% Tween-20 in PBS.

• Use alternative antibodies: If possible, test antibodies raised against different epitopes of TRAPPC2L - the immunogen region (amino acids 3-118 or the full sequence MAVCIAVIAKENYPLYIRSTPTENELKFHYMVHTSLDVVDEKISAMGKALVDQRELYLGLLYPTEDYKVYG) may affect specificity .

What are the optimal storage and handling conditions to maintain TRAPPC2L antibody performance?

To ensure maximum antibody performance and longevity:

• Storage temperature: Store antibodies at -20°C as recommended by manufacturers . Avoid repeated freeze-thaw cycles.

• Aliquoting: Upon receipt, divide the antibody into small working aliquots to minimize freeze-thaw cycles.

• Reconstitution: For lyophilized antibodies, reconstitute in 100 μl of sterile distilled H₂O with 50% glycerol as buffer .

• Working dilutions: Prepare fresh working dilutions on the day of use and store at 4°C for short-term use (1-2 weeks).

• Shipping conditions: When receiving antibodies, ensure they were shipped appropriately (wet ice for liquid antibodies) .

• Buffer composition: Note that commercial preparations may contain preservatives like 0.02% NaN₃ and stabilizers like 1% BSA .

Following these practices will help maintain antibody performance and extend shelf-life beyond the typical 12-18 months.

How can researchers effectively use TRAPPC2L antibodies to investigate TRAPP complex formation and dynamics?

For studying TRAPP complex dynamics:

• Co-immunoprecipitation (Co-IP): Use TRAPPC2L antibodies to pull down associated TRAPP complex components. This can reveal protein-protein interactions within the complex.

• Size exclusion chromatography: Combined with Western blotting using TRAPPC2L antibodies, this approach can distinguish different TRAPP complexes. Researchers have shown that variants in TRAPPC2L affect the assembly of TRAPP complexes, which can be detected by comparing chromatography profiles of wild-type versus mutant samples .

• Proximity ligation assays: These can detect in situ interactions between TRAPPC2L and other TRAPP components with spatial resolution.

• Immunofluorescence co-localization: Double labeling with TRAPPC2L antibody and antibodies against other TRAPP components (TRAPPC1, TRAPPC3, TRAPPC6a) can reveal their spatial relationships.

• FRAP (Fluorescence Recovery After Photobleaching): Combined with fluorescently-tagged TRAPPC2L and antibody validation, this technique can analyze the dynamics of TRAPP complex assembly and disassembly.

When interpreting results, remember that TRAPPC2L interacts with TRAPPC6a and may function as a putative adaptor for other TRAPP subunits, as demonstrated by yeast two-hybrid assays and in vitro binding studies .

What controls should be included when studying TRAPPC2L variants using antibody-based techniques?

When investigating TRAPPC2L variants (such as p.Ala2Gly or p.Asp37Tyr) using antibody-based approaches, include these controls:

• Wild-type TRAPPC2L: Essential baseline for comparative studies.

• Isogenic cell lines: Create cell lines with specific TRAPPC2L variants using CRISPR-Cas9 to isolate the effect of the mutation.

• Antibody validation controls:

  • Pre-absorption with recombinant TRAPPC2L protein to confirm specificity

  • TRAPPC2L knockdown/knockout samples to validate antibody specificity

  • Cross-reactivity testing with TRAPPC2 protein due to structural similarity

• Functional controls:

  • Membrane trafficking assays to assess functional consequences of variants

  • RAB11 activation assays, as TRAPPC2L variants have been shown to affect RAB11 activity

• Protein stability controls: CD spectroscopy and UV-visible spectroscopy can assess whether variants affect protein folding and stability, which may impact antibody recognition .

In published studies, researchers determined that the p.Ala2Gly variant, but not the p.Asp37Tyr variant, disrupted interaction between TRAPPC2L and TRAPPC6a, highlighting the importance of position-specific controls when studying variants .

How can TRAPPC2L antibodies be used to investigate the role of this protein in neurodevelopmental disorders?

To investigate TRAPPC2L's role in neurodevelopmental disorders:

• Tissue-specific expression profiling: Use TRAPPC2L antibodies to compare expression patterns in brain tissue from normal controls versus patients with neurodevelopmental disorders.

• Cellular model systems:

  • Create neuronal cell models expressing TRAPPC2L variants identified in patients (e.g., p.Ala2Gly, p.Asp37Tyr)

  • Use antibodies to track subcellular localization changes in mutant versus wild-type neurons

• Organoid studies: Apply TRAPPC2L antibodies in brain organoid models to investigate protein expression and localization in a three-dimensional context that better recapitulates human brain development.

• Pathway analysis: Use co-localization studies with markers of membrane trafficking pathways to determine which specific pathways are disrupted by TRAPPC2L mutations.

• RAB11 activity measurements: Since TRAPPC2L variants have been shown to increase levels of active RAB11 , use antibodies specific to active RAB11 alongside TRAPPC2L antibodies to correlate changes.

• Membrane trafficking assays: As demonstrated in patient fibroblasts, TRAPPC2L mutations cause delays in traffic into and out of the Golgi . Use TRAPPC2L antibodies in combination with trafficking markers to visualize these defects.

This multi-faceted approach can help elucidate how TRAPPC2L mutations contribute to neurodevelopmental phenotypes observed in affected individuals.

How do polyclonal and monoclonal TRAPPC2L antibodies compare for different research applications?

What methodological approaches can differentiate between TRAPPC2L and its structurally similar counterpart TRAPPC2?

Differentiating between TRAPPC2L and TRAPPC2 requires careful experimental design:

• Epitope selection: Choose antibodies raised against regions with minimal sequence homology between TRAPPC2L and TRAPPC2. The full TRAPPC2L immunogen sequence (MAVCIAVIAKENYPLYIRSTPTENELKFHYMVHTSLDVVDEKISAMGKALVDQRELYLGLLYPTEDYKVYG) differs from TRAPPC2, enabling specific detection .

• Validation methods:

  • Western blot analysis comparing recombinant TRAPPC2L and TRAPPC2

  • Immunoprecipitation followed by mass spectrometry to confirm target identity

  • Parallel knockdown experiments (siRNA against TRAPPC2L or TRAPPC2) to validate antibody specificity

• Functional assays: Based on the differential distribution of these proteins (TRAPPC2L associates with very low-density membrane fractions that do not contain Golgi markers), membrane fractionation followed by immunoblotting can distinguish their unique localization patterns .

• Cross-adsorption: Pre-adsorb TRAPPC2L antibodies with recombinant TRAPPC2 protein to remove cross-reactive antibodies.

• Double-labeling immunofluorescence: Use differently-labeled antibodies against TRAPPC2L and TRAPPC2 to visualize their distinct localization patterns within cells.

These approaches are crucial for studies investigating the non-redundant functions of these structurally similar proteins in membrane trafficking.

What are the most effective experimental designs to study TRAPPC2L interactions with other TRAPP complex components?

To study TRAPPC2L interactions with other TRAPP components:

• Yeast two-hybrid (Y2H) analysis: This approach has successfully demonstrated that the p.Ala2Gly variant in TRAPPC2L disrupts interaction with TRAPPC6a. The experimental setup involves:

  • Cloning TRAPP genes into pGADT7 and pGBKT7 plasmids

  • Transforming into AH109 and Y187 yeast strains respectively

  • Mating yeast on YPD plates at 30°C

  • Assessing interactions by growth on selective media (TDO plates lacking tryptophan and histidine)

• In vitro binding assays: These can quantitatively measure interactions using:

  • GST-tagged TRAPPC2L (wild-type and variants)

  • His-tagged binding partners (e.g., TRAPPC3-TRAPPC6a heterodimeric complex)

  • Increasing concentrations of binding partners (0-1.0 μM)

  • Detection via Western blotting with specific antibodies

• Size exclusion chromatography: This technique can reveal how TRAPPC2L variants affect TRAPP complex assembly. Fractions can be analyzed by Western blotting using antibodies against TRAPPC8, TRAPPC12, TRAPPC2L, and TRAPPC10 .

• FRET/BRET assays: These provide real-time, in-cell measurements of protein-protein interactions with high sensitivity.

• Proximity-dependent biotin identification (BioID): This approach can identify both stable and transient interactions of TRAPPC2L within the cellular environment.

These complementary approaches provide comprehensive insights into how TRAPPC2L functions as a putative adaptor for other TRAPP subunits and how disease-causing variants disrupt these interactions.

How might emerging technologies enhance TRAPPC2L antibody-based research?

Emerging technologies offer exciting possibilities for advancing TRAPPC2L research:

• Super-resolution microscopy: Techniques like STORM, PALM, and STED, combined with highly specific TRAPPC2L antibodies, can reveal the nanoscale organization of TRAPP complexes within membrane trafficking pathways with unprecedented detail.

• Expansion microscopy: This technique physically expands biological specimens, allowing conventional microscopes to resolve nanoscale structures when using TRAPPC2L antibodies for immunolabeling.

• Mass cytometry (CyTOF): Metal-conjugated TRAPPC2L antibodies can be used for highly multiplexed analysis of TRAPP complex components in single cells.

• Single-molecule tracking: When combined with photoactivatable fluorophore-conjugated antibodies or Fab fragments, this approach can reveal the dynamics of individual TRAPPC2L molecules in live cells.

• Spatial transcriptomics with protein detection: Combining TRAPPC2L antibody detection with spatial transcriptomics can correlate protein localization with gene expression patterns in tissues.

• Cryo-electron tomography with immunogold labeling: This technique can visualize TRAPPC2L within its native cellular environment at molecular resolution.

These technologies will help resolve longstanding questions about the precise role of TRAPPC2L in membrane trafficking and how its dysfunction contributes to human disease.

How can antibody-based approaches help elucidate the relationship between TRAPPC2L dysfunction and human disease?

Antibody-based approaches can significantly advance our understanding of TRAPPC2L in disease contexts:

• Patient-derived cell studies: TRAPPC2L antibodies can be used to analyze protein expression, localization, and complex formation in fibroblasts or induced pluripotent stem cells from patients with TRAPPC2L variants.

• Tissue microarrays: Applying TRAPPC2L antibodies to tissue microarrays containing samples from neurodevelopmental disorders can reveal expression patterns across multiple patients and conditions.

• Post-translational modification analysis: Antibodies specific to modified forms of TRAPPC2L can determine whether disease variants affect protein regulation through altered post-translational modifications.

• High-content screening: Automated microscopy with TRAPPC2L antibodies can assess the effects of therapeutic compounds on protein localization and trafficking defects in cellular disease models.

• In vivo disease models: TRAPPC2L antibodies can evaluate protein expression and distribution in animal models of neurodevelopmental disorders linked to TRAPPC2L mutations.

These approaches can bridge the gap between molecular dysfunction and clinical manifestations, potentially identifying therapeutic targets for TRAPPC2L-associated diseases.