TRAPPC3 Antibody

What is TRAPPC3 and what are its main cellular functions?

TRAPPC3 (also known as BET3) is a highly conserved core component of the TRAPP (Transport Protein Particle) complexes, which function as tethering factors in vesicular transport. It plays crucial roles in ER-to-Golgi transport and is involved in multiple membrane trafficking pathways. TRAPPC3 is the most conserved subunit of TRAPP and has been used to precipitate the intact tethering complex from both yeast and human cells . The protein is approximately 20 kDa with 180 amino acids and is localized primarily to the cis-Golgi membrane . Recent research has also shown that TRAPPC3 relocates to stress granules under oxidative stress conditions, suggesting additional roles beyond canonical membrane trafficking .

What types of TRAPPC3 antibodies are commercially available for research?

Multiple formats of TRAPPC3 antibodies are available, including:

• Polyclonal antibodies from rabbit and mouse hosts

• Antibodies targeting different epitopes (N-terminal, C-terminal, full-length)

• Unconjugated and conjugated (e.g., HRP-conjugated) formats

• Antibodies validated for various applications including Western blotting, immunohistochemistry, immunofluorescence, and immunoprecipitation

Which species do TRAPPC3 antibodies typically recognize?

Most commercially available TRAPPC3 antibodies recognize human TRAPPC3, with some cross-reactivity to mouse and rat orthologs. The high degree of evolutionary conservation in TRAPPC3 enables some antibodies to detect the protein across multiple mammalian species. For example, certain antibodies have been validated for human, mouse, and rat reactivity . When selecting an antibody for your research, it's important to verify species reactivity in the antibody documentation.

What are the optimal conditions for Western blotting with TRAPPC3 antibodies?

For Western blotting applications with TRAPPC3 antibodies:

• Recommended dilutions typically range from 1:500 to 1:3000, depending on the specific antibody

• More precisely, some manufacturers recommend 0.04-0.4 μg/mL for immunoblotting

• TRAPPC3 is typically observed at 20-22 kDa on Western blots

• Both reducing and non-reducing conditions are generally suitable

• Tissue and cell lysates showing robust TRAPPC3 expression include liver tissue, PC-3 cells, HEK-293 cells, and small intestine tissue

How should I optimize immunofluorescence experiments with TRAPPC3 antibodies?

For successful immunofluorescence detection of TRAPPC3:

• Typical dilutions range from 1:200 to 1:800 or 0.25-2 μg/mL

• HeLa cells show good expression levels for IF studies

• Under normal conditions, TRAPPC3 shows primarily Golgi/ER localization

• Under stress conditions (e.g., sodium arsenite treatment), TRAPPC3 relocates to stress granules and co-localizes with stress granule markers like eIF3

• When investigating TRAPPC3 in tissues like salivary glands, TRAPPC3 shows a bimodal distribution in the Golgi stack with concentration at both cis and trans sides

What controls should I include when validating a new TRAPPC3 antibody?

When validating a TRAPPC3 antibody, include the following controls:

• Positive control: Tissues/cells known to express TRAPPC3 (liver, HeLa, HEK293)

• Negative control: Secondary antibody only (no primary)

• Specificity control: TRAPPC3 knockdown or knockout cells/tissues

• For co-localization studies: Include markers for relevant cellular compartments (Golgi, ER, stress granules)

• If investigating complex formation: Compare with other TRAPP complex components (TRAPPC1, TRAPPC2, etc.)

How can I use TRAPPC3 antibodies to distinguish between TRAPPII and TRAPPIII complexes?

TRAPPC3 is present in both TRAPPII and TRAPPIII complexes, requiring strategic experimental design to distinguish between them:

• Co-immunoprecipitation approach:

  • Use TRAPPC3 antibodies to immunoprecipitate all TRAPP complexes

  • Analyze co-precipitating proteins by Western blot using complex-specific subunit antibodies:

    • TRAPPC9 and TRAPPC10 for TRAPPII-specific components

    • TRAPPC8 and TRAPPC11 for TRAPPIII-specific components

• Gradient fractionation approach:

  • Fractionate cell lysates on density gradients

  • Analyze fractions by Western blot using antibodies against:

    • TRAPPC3 (marks both complexes)

    • TRAPPC9/TRAPPC10 (TRAPPII-specific)

    • TRAPPC8/TRAPPC11 (TRAPPIII-specific)

• CRISPR/Cas9-mediated tagging:

  • Tag TRAPPC3 and complex-specific subunits as demonstrated in Drosophila studies

  • Use affinity purification followed by mass spectrometry to identify complex-specific interactors

What experimental approaches can detect TRAPPC3 relocation during cellular stress?

Research has shown that TRAPPC3 relocates to stress granules under stress conditions. To study this phenomenon:

• Stress induction protocols:

  • Oxidative stress: Treat cells with sodium arsenite (typically 0.5 mM for 30-60 minutes)

  • Heat shock: Expose cells to elevated temperatures (42-45°C for 30-60 minutes)

• Co-localization analysis:

  • Use TRAPPC3 antibodies alongside established stress granule markers (eIF3, G3BP, TIA-1)

  • Perform confocal microscopy to visualize co-localization

  • Quantify percentage of cells showing TRAPPC3-positive stress granules

• Mechanism investigation:

  • Use inhibitors of key pathways (such as CDK1/2 inhibitors) to examine factors affecting TRAPPC3 recruitment to stress granules

  • Apply siRNA knockdown of candidate regulatory proteins

How can TRAPPC3 antibodies be used to study its role in vesicular transport?

To investigate TRAPPC3's role in vesicular transport:

• TRAPPC3 knockout/knockdown approaches combined with antibody detection:

  • Generate TRAPPC3-deficient cells using CRISPR/Cas9 or RNAi

  • Use antibodies to confirm protein depletion

  • Examine effects on Rab1 localization (TRAPPIII acts as a Rab1 GEF)

  • Monitor ER-to-Golgi transport using trafficking assays

• In vitro GEF activity assays:

  • Immunoprecipitate TRAPP complexes using TRAPPC3 antibodies

  • Assess guanine nucleotide exchange activity on Rab1 and Rab11

  • Compare activity of TRAPPII and TRAPPIII complexes

• Proximity labeling approaches:

  • Combine TRAPPC3 antibodies with BioID or APEX techniques

  • Identify proteins in close proximity to TRAPPC3 under various conditions

Why might I observe multiple bands when using TRAPPC3 antibodies in Western blotting?

Multiple bands in Western blotting with TRAPPC3 antibodies can occur for several reasons:

• Isoform detection:

  • Human TRAPPC3 has multiple isoforms that may be detected by some antibodies

  • Cross-reactivity with other TRAPP complex members, particularly TRAPPC5/TRAPPC6, which are structural homologs of TRAPPC3

• Post-translational modifications:

  • TRAPPC3 can undergo palmitoylation, which may affect mobility on SDS-PAGE

  • Phosphorylation or other modifications might create additional bands

• Sample preparation issues:

  • Proteolytic degradation during preparation

  • Incomplete denaturation of protein complexes

Resolution approaches include using freshly prepared samples, adding additional protease inhibitors, optimizing antibody dilution, and comparing with knockdown controls.

How do mutations in TRAPPC3 affect antibody recognition and complex formation?

TRAPPC3 mutations can impact antibody binding and complex formation in several ways:

What factors should I consider when comparing TRAPPC3 localization across different cell types or tissues?

When comparing TRAPPC3 localization across different biological samples:

• Expression level variations:

  • TRAPPC3 expression levels differ across tissues and cell types

  • Adjust antibody concentrations accordingly for optimal signal-to-noise ratio

• Tissue-specific patterns:

  • TRAPPC3 shows distinct localization patterns in different tissues

  • In salivary glands, it displays a bimodal distribution in the Golgi stack

  • In some cell types, it may associate more with either cis or trans-Golgi elements

• Technical considerations:

  • Fixation methods can affect epitope accessibility

  • For tissues, antigen retrieval methods may be necessary (e.g., TE buffer pH 9.0 or citrate buffer pH 6.0)

  • Background autofluorescence varies by tissue type

• Co-localization standards:

  • Use consistent markers for cellular compartments across samples

  • Quantify co-localization using appropriate statistical methods

How can TRAPPC3 antibodies be used to study its role in stress responses beyond stress granules?

Recent research has revealed TRAPPC3's involvement in stress responses:

• Secretion arrest studies:

  • Use TRAPPC3 antibodies to track protein localization during various stresses

  • Monitor co-localization with secretory pathway markers during stress recovery

  • Combine with live-cell imaging of secretory cargo

• Stress signaling pathway investigation:

  • Examine TRAPPC3 phosphorylation status during stress using phospho-specific antibodies

  • Investigate interaction with CDK1/2 and other kinases implicated in stress responses

• Evolutionary conservation analysis:

  • Compare TRAPPC3 stress responses across species using cross-reactive antibodies

  • The stress response appears conserved from yeast to humans

What methodological advances are improving detection sensitivity for low-abundance TRAPPC3?

Several approaches can enhance detection sensitivity for TRAPPC3:

• Signal amplification techniques:

  • Tyramide signal amplification (TSA) for immunofluorescence

  • High-sensitivity chemiluminescent substrates for Western blotting

• Proximity ligation assays (PLA):

  • Detect TRAPPC3 interactions with other proteins with single-molecule sensitivity

  • Useful for tissues with low TRAPPC3 expression

• Mass spectrometry-based approaches:

  • Combine immunoprecipitation with highly sensitive mass spectrometry

  • Targeted approaches like selected reaction monitoring (SRM) for absolute quantification