TRAPPC11 Antibody

What is the molecular characterization of human TRAPPC11 protein?

TRAPPC11 (Trafficking Protein Particle Complex 11) is a component of the TRAPP III complex that functions in membrane trafficking and autophagy. The canonical human protein has 1133 amino acid residues with a molecular weight of approximately 129 kDa and is localized to the Golgi apparatus . TRAPPC11 contains a conserved "foie gras" domain important for its function, and participates in numerous interactions with other TRAPP complex components, including TRAPPC2, TRAPPC2L, TRAPPC6, TRAPPC10, and TRAPPC12 . Up to four different isoforms have been reported for this protein .

Which applications are TRAPPC11 antibodies validated for?

Current commercially available TRAPPC11 antibodies have been validated for multiple experimental applications:

Note: Optimization for each specific experimental system is strongly recommended .

Why is TRAPPC11 difficult to detect in certain experimental contexts?

TRAPPC11 can be challenging to analyze in fibroblasts due to limited antibody efficiency for detecting endogenous TRAPPC11 via western blotting . The protein's large size (129 kDa) and potential post-translational modifications can affect antibody recognition. Studies have shown that using higher concentrations of antibody (1:500) and optimizing protein extraction protocols with phosphatase inhibitors can improve detection . Some researchers have addressed this limitation by using tagged TRAPPC11 constructs for overexpression studies.

How can TRAPPC11 antibodies be used to study trafficking defects in patient cells?

To assess membrane trafficking defects associated with TRAPPC11 mutations, researchers can employ:

• ER-to-Golgi trafficking assay: Using Retention Using Selective Hooks (RUSH) assay with TRAPPC11 antibody co-staining to quantify transport kinetics .

• Golgi apparatus architecture analysis: Immunostaining with TRAPPC11 antibody alongside Golgi marker protein GM130. Patient fibroblasts with TRAPPC11 mutations typically display punctate Golgi dispersal similar to that observed after TRAPPC11 knockdown in HeLa cells .

• Protein trafficking measurement: Track the transport of secretory proteins from ER through Golgi to cell surface using a combination of TRAPPC11 and secretory protein antibodies, with analysis by confocal microscopy and time-lapse imaging .

What experimental protocol is recommended for assessing mitochondrial function in TRAPPC11-deficient cells?

Recent studies have identified a novel role for TRAPPC11 in mitochondrial function. The following protocol has been validated:

• Non-glycolytic ATP production measurement:

  • Incubate cells from individuals with TRAPPC11-related muscular dystrophy and control cells with 2-deoxy-D-glucose for 2 hours

  • Assay ATP concentration by bioluminescence using a luciferin-luciferase system

  • Correct measurements by protein concentration and express as fold change over controls

  • Perform experiments at least three times with triplicates

• Mitochondrial network visualization:

  • Conduct immunofluorescence against TOMM20 (1:250, ab186735)

  • Image using confocal microscopy (e.g., Zeiss LSM 880) with 63× magnification

  • Analyze images with Fiji software and parametrize using Mitochondrial Analyzer plugin

  • Compare mitochondrial number, branch length, and network complexity between patient and control cells

How can researchers characterize the phenotypic spectrum of TRAPPC11 mutations in patient samples?

Characterization of TRAPPC11-related pathologies requires a comprehensive approach:

• Clinical assessment: Document early-onset muscle weakness, movement disorders, intellectual disability, elevated serum creatine kinase, microcephaly, and infection-triggered psychomotor regression .

• Muscle biopsy analysis:

  • Process samples following standard histological, histochemical, and immunohistochemical protocols

  • Look for variation in fiber size, atrophic fibers, regenerating fibers, internally placed nuclei, and increased connective tissue

  • Perform immunohistochemistry for sarcolemmal proteins (α-, β-, γ-, δ-sarcoglycan)

• Western blot analysis:

  • Analyze TRAPPC11 expression levels in fibroblast lysates

  • Assess α-dystroglycan expression (using VIA4 and IIH6 antibodies)

  • Compare band intensity and molecular weight between patient and control samples

  • Look for reduction in α-dystroglycan expression in some patients, which can be subtle to moderate

• In silico analysis:

  • Construct homology molecular models based on TRAPP complex structures

  • Visualize human TRAPPC11 structural models using tools like PyMOL

  • Analyze the potential impact of mutations on protein structure and function

What is the recommended approach for studying the effect of TRAPPC11 mutations on autophagy?

Autophagy defects are a key feature in some TRAPPC11 mutations. The following protocol has been validated:

• Autophagy flux assessment:

  • Measure LC3-II levels by western blot in patient and control cells

  • Normalize LC3-II signal to loading controls like tubulin

  • Quantify using image analysis software (e.g., ImageJ)

  • Perform statistical analysis using two-way ANOVA with post-hoc Tukey HSD

• Therapeutic exploration:

  • For nonsense mutations, test translational read-through drugs (TRIDs) like Ataluren and Amlexanox

  • Evaluate rescue of autophagy defects by measuring changes in LC3-II levels

  • Note that response may vary by mutation type (compound heterozygous mutations may show different responses than homozygous mutations)

How can researchers distinguish between TRAPPC11 roles in trafficking versus glycosylation defects?

TRAPPC11 has dual functions that can be experimentally separated:

• Trafficking-specific assays:

  • Measure ER-to-Golgi transport rates using RUSH assay

  • Assess protein exit from Golgi to cell surface

  • Analyze Golgi architecture using GM130 staining

• Glycosylation-specific assays:

  • Analyze N-linked glycosylation using glycoprotein detection methods

  • Measure lipid-linked oligosaccharide (LLO) levels

  • Assess expression of genes in the terpenoid biosynthetic pathway

  • Treat wild-type samples with terpenoid or LLO synthesis inhibitors to phenocopy glycosylation defects

• Comparative analysis:

  • Compare TRAPPC11 depletion effects to depletion of other TRAPP components

  • Unlike other TRAPP components, only TRAPPC11 depletion causes protein hypoglycosylation in human cells

  • Western blot analysis of α-dystroglycan can reveal glycosylation defects specific to TRAPPC11 dysfunction

What methodological approaches are recommended for investigating potential digenic inheritance involving TRAPPC11?

Recent studies have identified potential digenic inheritance patterns involving TRAPPC11. The following methodological approach is recommended:

• Genetic screening:

  • Perform whole exome sequencing and Sanger sequencing validation

  • Design locus-specific PCR primers for potential variants (e.g., TRAPPC11 variant: 5′-AAGCCATAAGTGGGGAGCTA-3′ and 5′-ATCACTGGGCTCCACAGAAA-3′)

  • Analyze conservation using BLAST

• Protein interaction analysis:

  • Use tools like GeneMANIA to identify potential interactions between candidate genes

  • TRAPPC11 has been found to be co-expressed with genes that physically interact with TTN in LGMD cases

  • This approach can reveal potential shared pathogenic pathways involving TRAPPC11 and other proteins

• Functional validation:

  • Evaluate phenotypic manifestations in carriers of single vs. digenic mutations

  • Examine muscle biopsy findings for specific pathological signatures

  • Investigate synergistic effects through cell culture models

What controls are essential when working with TRAPPC11 antibodies?

Proper controls are critical for reliable TRAPPC11 research:

• Positive controls:

  • HeLa cells and U2OS cells express detectable levels of endogenous TRAPPC11

  • Mouse and rat brain tissues also serve as reliable positive controls for western blotting

• Negative controls:

  • TRAPPC11 siRNA/shRNA knockdown cells

  • Isotype control antibodies

  • Secondary antibody-only controls for immunostaining

• Special considerations:

  • For IHC applications, antigen retrieval with TE buffer pH 9.0 is recommended

  • Alternatively, citrate buffer pH 6.0 may be used for antigen retrieval

  • Use cells between passages 7 and 12 for consistent results

How should researchers interpret contradictory findings regarding mitochondrial versus Golgi defects in TRAPPC11 mutations?

Recent studies have revealed expanded roles for TRAPPC11 beyond its canonical Golgi function:

• Mechanistic framework:

  • TRAPPC11 likely functions within a mitochondria-Golgi-ER triad

  • Different mutations may affect distinct protein domains and functions

  • The foie gras domain (affected by p.Ala372_Ser429del mutation) plays a role in both trafficking and potentially mitochondrial function

• Experimental approach to resolve contradictions:

  • Perform domain-specific mutations and assess differential effects

  • Conduct rescue experiments with wild-type TRAPPC11 to determine which phenotypes can be reversed

  • Implement tissue-specific analyses, as some effects may be cell-type dependent

  • Use time-course experiments to distinguish primary from secondary effects

• Integrated interpretation:

  • Some mitochondrial defects may be secondary consequences of primary Golgi/trafficking defects

  • Different mutations may preferentially affect distinct TRAPPC11 functions

  • The stressed UPR observed in some models may link the seemingly disparate phenotypes

What emerging research areas are most promising for TRAPPC11 antibody applications?

Several cutting-edge research directions show particular promise:

• Therapeutic development:

  • Translational read-through drugs (TRIDs) for nonsense mutations

  • Targeted therapies addressing specific cellular defects (glycosylation, trafficking, mitochondrial)

  • Understanding variant-specific responses to potential treatments

• Multi-omics integration:

  • Combining proteomic, transcriptomic, and metabolomic approaches to understand TRAPPC11 deficiency

  • Investigating tissue-specific effects of mutations

  • Exploring the relationship between TRAPPC11 and other muscular dystrophy-associated proteins

• Developmental biology applications:

  • Investigating TRAPPC11's role in myeloid cell development and differentiation

  • Understanding the basis for microcephaly in TRAPPC11-related disorders

  • Exploring potential roles in embryonic development using model systems

• Expanded phenotypic spectrum:

  • Using TRAPPC11 antibodies to investigate potential roles in unexplored tissues

  • Examining connections to other organelle dysfunctions beyond Golgi and mitochondria

  • Investigating the basis for metabolic decompensation during infections in patients