TRAPPC6B Antibody

What is TRAPPC6B and what are its known cellular functions?

TRAPPC6B (Trafficking Protein Particle Complex Subunit 6B) is a component of the TRAPP complex that functions in specific stages of inter-organelle traffic. The protein plays a critical role in vesicle-mediated transport within cells . Research indicates that TRAPPC6B is specifically involved in the early development of neural circuitry, likely by controlling the frequency and amplitude of intracellular calcium transients that regulate neuron differentiation and survival . This 18 kDa protein is encoded by a gene located on chromosome 14q21.1 in humans .

What applications are TRAPPC6B antibodies validated for?

TRAPPC6B antibodies have been validated for multiple research applications:

Successful visualization has been demonstrated in human adrenal gland, pancreas, and mouse kidney tissues . When performing Western blot, researchers should expect to observe bands at approximately 18 kDa, which corresponds to the predicted molecular weight of TRAPPC6B .

How should TRAPPC6B antibodies be stored and handled?

For optimal antibody performance and longevity:

• Store at -20°C in aliquots to minimize freeze-thaw cycles

• Most commercial preparations contain PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Antibodies remain stable for approximately one year after shipment when stored properly

• Smaller size formats (e.g., 20μL) may contain 0.1% BSA as a stabilizer

• According to manufacturer data, aliquoting may not be necessary for -20°C storage

Proper storage conditions are critical for maintaining antibody specificity and sensitivity across experiments.

What positive and negative controls should be used with TRAPPC6B antibodies?

Rigorous experimental design requires appropriate controls:

Positive controls:

• Mouse kidney tissue lysate shows detectable TRAPPC6B expression in Western blots

• Human adrenal gland and pancreas tissues demonstrate positive staining in IHC applications

Negative controls:

• Primary antibody omission control

• Isotype control (using rabbit IgG at equivalent concentration)

• RNAse R-treated samples can serve as controls to distinguish between circular and linear forms of TRAPPC6B RNA

For Western blot applications, β-actin is commonly used as a loading control to normalize TRAPPC6B expression levels .

How can researchers validate the specificity of TRAPPC6B antibodies?

Comprehensive antibody validation strategies include:

• Epitope verification: Confirm the immunogen details - many commercial antibodies target specific regions (e.g., amino acids 1-150 of human TRAPPC6B)

• Cross-validation approaches:

  • Compare results using antibodies targeting different TRAPPC6B epitopes

  • Validate with orthogonal methods like RT-PCR or in situ hybridization

  • Pre-absorption tests with immunizing peptides to confirm specificity

• Genetic validation:

  • CRISPR/Cas9 knockout or siRNA knockdown followed by antibody testing

  • Overexpression systems to confirm signal increases

• Species cross-reactivity assessment: Test antibody performance across multiple relevant species according to experimental design (most commercial antibodies react with human, mouse, and rat samples)

What is the relationship between circTRAPPC6B and linear TRAPPC6B?

CircTRAPPC6B is a circular RNA derived from the TRAPPC6B gene with distinct properties from its linear counterpart:

• Structural differences:

  • CircTRAPPC6B forms through back-splicing of exons 3 and 4 of the TRAPPC6B gene

  • The mature circTRAPPC6B is 202 bp in length

  • Unlike linear RNA, it lacks 5' and 3' ends, forming a closed loop structure

• Experimental differentiation methods:

  • RNase R treatment: CircRNAs resist digestion while linear RNAs degrade

  • RT-PCR with divergent primers targeting the back-splice junction

  • FISH analysis with junction-specific probes

• Functional differences:

  • CircTRAPPC6B functions as a miRNA sponge, specifically targeting miR-874-3p

  • This function differs from the protein-coding role of linear TRAPPC6B mRNA

When studying TRAPPC6B, researchers must carefully design experiments that distinguish between these forms to avoid confounding results.

How does circTRAPPC6B influence autophagy in Mycobacterium tuberculosis infection?

CircTRAPPC6B plays a critical role in regulating autophagy during Mycobacterium tuberculosis (Mtb) infection through a well-characterized molecular mechanism:

• Autophagy induction pathway:

  • CircTRAPPC6B enhances autophagy in Mtb-infected macrophages

  • It increases autophagy-associated protein LC3-II production

  • Enhanced autophagy aggregation is observable via fluorescence in situ hybridization

• Molecular mechanism (miRNA regulatory circuit):

  • CircTRAPPC6B acts as a miRNA sponge, sequestering miR-874-3p

  • miR-874-3p normally suppresses autophagy by binding to ATG16L1's 3'-UTR

  • By inhibiting miR-874-3p, circTRAPPC6B enhances ATG16L1 expression

  • Increased ATG16L1 activates autophagy pathways that restrict Mtb growth

• Expression dynamics:

  • CircTRAPPC6B is significantly downregulated in PBMCs of patients with active TB

  • Expression recovers after one month of standardized anti-TB treatment

  • This pattern suggests circTRAPPC6B normally functions in antimycobacterial defense

This research highlights circTRAPPC6B as a potential therapeutic target for tuberculosis control.

What sample preparation methods optimize TRAPPC6B detection in different experimental contexts?

Sample preparation significantly impacts TRAPPC6B detection success:

• For Western blot analysis:

  • Extract proteins using RIPA buffer supplemented with protease inhibitors

  • Determine protein concentration via Bradford assay

  • Process samples (20 μg) with LDS buffer and reducing agent

  • Use 4-12% gradient gels with MOPS running buffer

  • Transfer to PVDF membrane with buffer containing 10% methanol and antioxidant

• For immunohistochemistry:

  • Perfusion-fix tissues followed by overnight fixation in 4% PFA/PBS

  • For brain tissues: dehydrate in 30% sucrose/PBS before cryosectioning (12 μm)

  • For other tissues: use paraffin embedding and sectioning (7 μm)

  • Critical antigen retrieval step: incubate sections in 10 mM Na-citrate buffer at 65°C

  • Quench endogenous peroxidase activity with methanol/0.3% H₂O₂

• For circular RNA analysis:

  • Treat samples with RNase R to digest linear RNAs, preserving circTRAPPC6B

  • RNAscope technology enables in situ detection in tissues

  • Use random hexamer primers and appropriate reverse transcriptase for RT-PCR

These optimized protocols have been validated in published research and provide reliable detection across experimental systems.

What is known about TRAPPC6B in neurodevelopmental contexts?

Research indicates important roles for TRAPPC6B and related TRAPP complex components in neurodevelopment:

• Microcephaly connection:

  • Knockout of Trappc9 (related to TRAPPC6B in the TRAPP complex) in mice results in postnatal-onset microcephaly

  • The condition is established by weaning age and includes disproportionate reduction in hippocampal size

  • This suggests TRAPP complex components are critical for proper brain development

• Neural stem cell implications:

  • Studies have examined Sox2-positive neural stem cells in the dentate gyrus in relation to TRAPP complex dysfunction

  • Differences in Sox2-positive cell counts were observed between wild-type and knockout models

  • This indicates potential roles in neural stem cell maintenance or differentiation

• Calcium signaling regulation:

  • TRAPPC6B likely functions by controlling intracellular calcium transients

  • These calcium signals regulate neuron differentiation and survival

  • Disruptions to this signaling may contribute to neurodevelopmental abnormalities

How can researchers troubleshoot weak TRAPPC6B detection in Western blot applications?

When encountering weak or absent TRAPPC6B signals, consider these methodical troubleshooting approaches:

• Sample preparation optimization:

  • Ensure complete protein extraction with fresh RIPA buffer containing protease inhibitors

  • Verify protein concentration with Bradford assay

  • Load adequate protein amount (≥20 μg total protein)

  • Use fresh reducing agents to ensure complete denaturation

  • Avoid sample degradation by minimizing freeze-thaw cycles

• Technical parameters adjustment:

  • Test a range of primary antibody dilutions (e.g., 1:500 to 1:2000)

  • Extend primary antibody incubation to overnight at 4°C

  • Compare blocking reagents (BSA vs. commercial blockers like Odyssey)

  • Verify transfer efficiency with Ponceau S staining

  • Consider switching between HRP and fluorescent detection systems

• TRAPPC6B-specific considerations:

  • Verify you're examining the correct molecular weight region (18 kDa)

  • Use tissues known to express TRAPPC6B as positive controls (kidney)

  • Consider possible post-translational modifications affecting detection

  • Test alternative antibodies targeting different epitopes within TRAPPC6B

A systematic approach to troubleshooting will help identify the specific factors limiting detection.

What experimental approaches can differentiate between functional effects of circular versus linear TRAPPC6B?

To distinguish between the roles of circular and linear TRAPPC6B forms:

• Selective knockdown strategies:

  • Design siRNAs targeting the back-splice junction to specifically reduce circTRAPPC6B

  • Use siRNAs targeting linear-specific regions to reduce only the linear form

  • CRISPR-Cas9 editing of splice sites can also selectively affect circular or linear forms

• Overexpression approaches:

  • Express circTRAPPC6B using specialized vectors that promote back-splicing

  • Compare to linear TRAPPC6B overexpression systems

  • Assess differential effects on cell processes like autophagy and vesicular transport

• miRNA interaction studies:

  • Test miR-874-3p binding specificity to circTRAPPC6B versus linear TRAPPC6B

  • Use pull-down assays and luciferase reporter systems to confirm interactions

  • Assess downstream effects on targets like ATG16L1

• Differential expression analysis:

  • Compare expression patterns of circular versus linear forms in different tissues

  • Assess changes during development or disease progression

  • Correlation with specific cellular functions

These approaches enable precise attribution of biological effects to the specific TRAPPC6B form.

How can TRAPPC6B antibodies be used to investigate its role in infectious disease contexts?

TRAPPC6B antibodies can elucidate host-pathogen interactions, particularly in tuberculosis research:

• Infection-induced expression changes:

  • Western blot analysis of TRAPPC6B protein levels pre- and post-infection

  • IHC to visualize changes in subcellular localization during infection

  • Correlation with circTRAPPC6B levels to understand RNA-protein relationships

• Autophagy pathway investigation:

  • Co-immunoprecipitation to identify TRAPPC6B interaction partners during infection

  • Co-localization studies with autophagy markers like LC3-II

  • Comparative analysis between wild-type and ATG16L1-deficient backgrounds

• Translational applications:

  • Assessment of TRAPPC6B as a biomarker for TB diagnosis

  • Correlation of protein levels with treatment response

  • Investigation of TRAPPC6B manipulation as a therapeutic strategy

• Experimental design considerations:

  • Include appropriate controls (uninfected cells, heat-killed bacteria)

  • Monitor time-course changes in expression and localization

  • Compare effects across different pathogens to assess specificity

This research direction has significant potential for identifying novel host-directed therapeutic targets for infectious diseases.