TECPR2 Antibody, Biotin conjugated

What is TECPR2 and why is it important in cellular function?

TECPR2 is a large multi-domain protein (1411 amino acids in humans) comprised of an N-terminal WD domain, a middle unstructured region, and a C-terminal TECPR domain containing six TECPR repeats followed by a functional LC3-interacting region (LIR) motif. This protein has gained significant attention due to its multifaceted roles in:

• Autophagy regulation, particularly in lysosomal targeting of autophagosomes

• Secretory pathway function and ER-Golgi transport

• Endosomal cargo recycling as a Rab5 effector

• Neuronal function, with mutations linked to hereditary sensory and autonomic neuropathy (HSAN)/spastic paraplegia type 49 (SPG49)

The importance of TECPR2 is underscored by the severity of neurodegenerative phenotypes observed when the protein is dysfunctional, making it a valuable target for research into cellular quality control mechanisms and neurodegeneration.

What are the key specifications of commercially available TECPR2 antibodies, Biotin conjugated?

Commercially available biotin-conjugated TECPR2 antibodies typically have the following specifications:

These antibodies are specifically designed for research use only and should not be used in diagnostic, therapeutic, or cosmetic procedures .

How should TECPR2 antibodies be stored and handled for optimal performance?

For maintaining optimal antibody performance in experimental applications:

• Temperature: Store at -20°C in small aliquots to minimize freeze-thaw cycles. Some manufacturers recommend storage at -80°C as an alternative .

• Light exposure: Biotin-conjugated antibodies are particularly sensitive to light; store in amber tubes or wrapped in foil.

• Reconstitution: If lyophilized, reconstitute only with recommended buffers and at the suggested concentration.

• Working solutions: Prepare fresh dilutions on the day of experiment rather than storing diluted antibody.

• Contamination prevention: Use sterile techniques when handling antibody solutions to prevent microbial growth.

• Centrifugation: Briefly centrifuge vials before opening to collect liquid at the bottom.

• Transport: When removing from freezer, transport on ice and return to storage promptly.

Proper storage and handling significantly impact experimental reproducibility, particularly in sensitive applications like immunofluorescence or co-immunoprecipitation studies with TECPR2 .

What cellular compartments does TECPR2 localize to, and how can the antibody be used to study this localization?

TECPR2 exhibits a complex subcellular distribution pattern that reflects its diverse functions:

• ER and ER exit sites (ERES): TECPR2 regulates the stability of COPII coat subunits (SEC24D-SEC23) and influences ER-to-Golgi transport .

• Early endosomes: As a Rab5 effector, TECPR2 localizes to early endosomes in a GTP-dependent manner through its C-terminal TECPR repeats .

• Autophagosomes: TECPR2 associates with autophagosomes via interaction with LC3B and other ATG8 family proteins through its LIR motif .

• Lysosomes: TECPR2 is recruited to lysosomes through interaction with VAMP8, playing a role in autophagosome-lysosome fusion .

To study this localization pattern using biotin-conjugated TECPR2 antibodies:

• Immunofluorescence approach:

  • Fix cells using 4% paraformaldehyde (10 minutes at room temperature)

  • Permeabilize with 0.1% Triton X-100 (5 minutes)

  • Block with 3% BSA in PBS (1 hour)

  • Incubate with biotin-conjugated TECPR2 antibody (optimal dilution determined empirically)

  • Detect using streptavidin-conjugated fluorophores

  • Co-stain with markers of specific compartments:

    • ER/ERES: SEC12, SEC16A, SEC23A, SEC24C

    • Early endosomes: Rab5, EEA1

    • Autophagosomes: LC3B

    • Lysosomes: LAMP1, LAMP2

• Subcellular fractionation:

  • Homogenize cells or tissues in appropriate buffer

  • Fractionate using sucrose gradient centrifugation

  • Analyze fractions by Western blotting using the biotin-conjugated TECPR2 antibody

  • Compare distribution with compartment-specific markers

Disease-associated TECPR2 mutations often disrupt normal localization patterns, providing valuable insights into protein function .

How can TECPR2 antibodies be used to investigate autophagy dysfunction in neurodegenerative disease models?

TECPR2 antibodies are valuable tools for investigating autophagy dysfunction in neurodegenerative disease models, particularly SPG49/HSAN9. Methodological approaches include:

• Autophagosome accumulation analysis:

  • Western blot analysis of brain lysates or patient-derived fibroblasts using TECPR2 antibodies in parallel with autophagy markers (LC3B, SQSTM1/p62)

  • Quantification of LC3B-II/LC3B-I ratio and SQSTM1/p62 levels as indicators of autophagy flux

  • Comparison between wild-type and disease models (TECPR2 knockout or patient-derived cells)

• Transmission electron microscopy (TEM) correlation:

  • TEM analysis of affected tissues to visualize accumulated autophagosomes

  • Immunogold labeling using biotin-conjugated TECPR2 antibody and streptavidin-gold

  • Quantification of autophagosome numbers, size, and distribution

• Autophagy flux assessment:

  • Treatment with bafilomycin A1 to block autophagosome-lysosome fusion

  • Measurement of LC3B-II accumulation in the presence vs. absence of treatment

  • Comparison between wild-type and diseased samples using TECPR2 antibodies for correlation

• Tandem fluorescent reporter assay:

  • Transfection with mRFP-GFP-LC3B construct

  • Identification of autophagosomes (yellow) vs. autolysosomes (red)

  • Correlation with TECPR2 expression and localization using antibody detection

• Rescue experiments:

  • Reintroduction of wild-type TECPR2 into mutant cells

  • Assessment of autophagy restoration using TECPR2 antibodies

  • Comparison with disease-associated mutants (e.g., L440Rfs)

These approaches have revealed that TECPR2 mutations lead to autophagosome accumulation, indicating impaired autophagosome-lysosome fusion in neurodegenerative conditions .

How can TECPR2 antibodies be used to study protein-protein interactions in autophagy pathways?

TECPR2 participates in multiple protein-protein interactions that regulate autophagy. Biotin-conjugated TECPR2 antibodies can be employed in several sophisticated approaches to investigate these interactions:

• Co-immunoprecipitation with streptavidin pulldown:

  • Lyse cells in mild buffer (1% NP-40, 150mM NaCl, 50mM Tris pH 7.5)

  • Incubate lysates with biotin-conjugated TECPR2 antibody

  • Precipitate using streptavidin-coated beads

  • Analyze precipitates for known TECPR2 interactors:

    • ATG8 family proteins (LC3B)

    • HOPS complex components (VPS41, VPS39, VPS33a, VPS16)

    • Lysosomal SNARE protein VAMP8

• Proximity-dependent biotinylation approaches:

  • Express APEX2-tagged TECPR2 or its interaction partners

  • Induce biotinylation in living cells

  • Enrich biotinylated proteins

  • Compare with conventional pulldown using biotin-conjugated TECPR2 antibodies

• Domain-specific interaction mapping:

  • Express distinct domains of TECPR2:

    • N-terminal WD domain

    • Middle unstructured region

    • C-terminal TECPR domain with LIR motif

  • Perform domain-specific pulldowns

  • Compare interaction profiles between domains

• LIR motif dependency assessment:

  • Compare interactions between wild-type TECPR2 and LIR mutants

  • Analyze effects on binding to ATG8 family proteins

  • Correlate with functional outcomes in autophagy assays

• HOPS complex association studies:

  • Investigate TECPR2's role in autophagosome-lysosome tethering

  • Determine whether TECPR2 bridges between Atg8 proteins and the VAMP8-HOPS complex

  • Assess effects of disease mutations on these interactions

These approaches have revealed that TECPR2 serves as a molecular bridge between autophagosomes and lysosomes, with its C-terminal domain playing a crucial role in these interactions .

What is the role of TECPR2 as a Rab5 effector, and how can antibodies be used to study this function?

Recent research has identified TECPR2 as an effector of the early endosomal small GTPase Rab5, representing a novel function beyond its established roles in autophagy. To investigate this function using TECPR2 antibodies:

• GTP-dependent binding assays:

  • Express and purify wild-type Rab5 and its mutants:

    • Constitutively GTP-bound (Q79L)

    • Constitutively GDP-bound (S34N)

  • Perform pulldown assays with recombinant TECPR2 or cellular lysates

  • Use biotin-conjugated TECPR2 antibodies to detect binding specificity

  • Confirm GTP-dependency of the interaction

• Mapping the Rab5 binding interface:

  • The C-terminal TECPR repeats (935-1411 a.a.) are both necessary and sufficient for Rab5 binding

  • Key residues include E1299, which when mutated to alanine disrupts Rab5 binding

  • Disease-associated mutations (W1140G, R1336W) impair Rab5 binding

  • Use antibodies to detect wild-type vs. mutant TECPR2 localization

• Membrane recruitment visualization:

  • Co-express Rab5 (wild-type, Q79L, or S34N) with TECPR2

  • Use immunofluorescence with TECPR2 antibodies to assess membrane recruitment

  • Compare wild-type TECPR2 with binding-defective mutants (E1299A, R1336W)

  • Validate findings with in vitro reconstitution using giant unilamellar vesicles (GUVs)

• Impact on endosomal recycling:

  • TECPR2 regulates the recycling of cargo such as α5β1 integrin receptors

  • TECPR2 affects the early endosomal localization of:

    • Cargo adaptor SNX17

    • WASH complex (mediates actin polymerization)

    • ARP2/3 complex

  • Use TECPR2 antibodies in combination with these markers to assess functional impacts

• Endosomal fractionation:

  • Purify early endosomes using established protocols

  • Compare protein composition in control vs. TECPR2-depleted conditions

  • Analyze impact on endosomal recruitment of cargo recycling machinery

This newly discovered function connects TECPR2 to endosomal cargo recycling and provides insights into how its dysfunction may contribute to neurodegenerative disorders through multiple cellular pathways .

How can I validate the specificity of TECPR2 antibodies in experimental applications?

Rigorous validation is essential for ensuring the reliability of results obtained using TECPR2 antibodies. Comprehensive validation strategies include:

• Genetic validation approaches:

  • Negative controls: Test antibody reactivity in TECPR2 knockout cells/tissues or after siRNA-mediated knockdown

  • Positive controls: Compare with cells overexpressing tagged TECPR2

  • Rescue experiments: Reintroduce wild-type TECPR2 into knockout backgrounds

  • Mutation analysis: Test antibody reactivity against disease-associated TECPR2 variants

• Domain-specific validation:

  • Express individual domains (N-terminal WD domain vs. C-terminal TECPR domain)

  • Determine epitope specificity using truncation constructs

  • For the biotin-conjugated antibodies available commercially, verify recognition of the immunogen region (516-783 AA)

• Application-specific validation:

  • Western blotting: Confirm single band of expected molecular weight (~141 kDa for full-length human TECPR2)

  • Immunofluorescence: Verify expected subcellular distribution pattern (ER, endosomes, autophagosomes)

  • ELISA: Establish standard curves using recombinant TECPR2 protein

  • IP: Confirm enrichment of known TECPR2 interaction partners

• Cross-reactivity assessment:

  • Test reactivity against related proteins (e.g., TECPR1, the closest homolog)

  • Verify species specificity (commercial antibodies are typically validated for human TECPR2)

  • Check for non-specific binding in various cell types and tissues

• Functional correlation:

  • Verify that antibody-detected changes correlate with functional outcomes

  • For autophagy studies, correlate with LC3B and p62 dynamics

  • For secretory pathway studies, correlate with ER exit site markers

  • For endosomal studies, correlate with cargo recycling phenotypes

Thorough validation ensures confidence in experimental findings and is particularly important when studying a multifunctional protein like TECPR2, which operates in multiple cellular compartments and pathways.

What technical considerations should be made when using biotin-conjugated antibodies in multiplex imaging experiments?

When designing multiplex imaging experiments with biotin-conjugated TECPR2 antibodies, several technical considerations are essential:

• Endogenous biotin interference:

  • Pre-block endogenous biotin using avidin/streptavidin blocking kits

  • Consider sequential detection strategies to minimize background

  • Include controls to assess endogenous biotin levels in your specific cell/tissue type

• Detection strategy optimization:

  • Select appropriate streptavidin conjugates (fluorophores, enzymes) based on experimental needs

  • Consider signal amplification methods for low-abundance targets:

    • Tyramide signal amplification (TSA)

    • Poly-HRP streptavidin

    • Quantum dot-conjugated streptavidin for enhanced brightness and stability

• Multiplexing considerations:

  • Plan staining sequence carefully when combining with other antibodies

  • If using multiple biotinylated primary antibodies, employ sequential detection with blocking steps

  • Consider spectral unmixing for fluorescence applications to separate overlapping signals

  • For brightfield applications, use different chromogens for separate detection

• Fixation and antigen retrieval optimization:

  • Test different fixation methods (paraformaldehyde vs. methanol) for TECPR2 detection

  • Optimize antigen retrieval conditions if working with fixed tissues

  • Ensure fixation method preserves biotin conjugation and target epitope accessibility

• Controls for multiplexed experiments:

  • Single-stain controls to establish spectral profiles

  • Secondary-only controls to assess non-specific binding

  • Biological controls (TECPR2 knockout, siRNA) to confirm specificity

  • Competition controls with unconjugated antibody

• Imaging parameters:

  • Adjust exposure settings to accommodate range of signal intensities

  • Consider photobleaching characteristics when designing acquisition protocols

  • Use consistent imaging parameters across experimental conditions for quantitative comparisons

These considerations will help ensure optimal results when using biotin-conjugated TECPR2 antibodies in conjunction with other detection reagents for multiplexed imaging applications.

How do mutations in TECPR2 contribute to neurodegenerative disorders, and how can antibodies help study these mechanisms?

TECPR2 mutations have been identified as the genetic basis for spastic paraplegia type 49 (SPG49)/hereditary sensory and autonomic neuropathy (HSAN9), providing insights into the protein's role in neuronal function. Research using TECPR2 antibodies has revealed multiple pathogenic mechanisms:

• Autophagy dysfunction:

  • TECPR2 mutations lead to impaired autophagosome-lysosome fusion

  • Electron microscopy of tecpr2-/- mouse brain reveals autophagosome accumulation in axonal spheroids

  • Patient fibroblasts show reduced colocalization of autophagosomes (LC3B-positive) and lysosomes (LAMP1-positive)

  • TECPR2 antibodies help visualize these defects through immunofluorescence studies

• Secretory pathway disturbances:

  • TECPR2 deficiency causes significantly decreased numbers of SEC24C- and SEC13-positive puncta

  • This indicates reduced functional ER exit sites (ERES)

  • TECPR2 antibodies enable visualization of these ERES components and their colocalization with TECPR2

• Endosomal recycling defects:

  • Recently discovered role as a Rab5 effector links TECPR2 to endosomal cargo recycling

  • Disease-associated variants (R1336W) show impaired Rab5 binding and membrane recruitment

  • Resulting in altered endosomal cargo sorting and recycling

  • TECPR2 antibodies help characterize these defects in patient-derived cells

• Molecular mechanism correlation with disease variants:

  • Nonsense mutations (c.1319delT, c.3416delT) lead to premature stop codons

  • Missense variants (T189I in WD domain, R1336W in TECPR domain) affect specific functions

  • TECPR2 antibodies help determine the impact of mutations on protein expression, stability, and localization

• Animal model validation:

  • Tecpr2 knockout mice exhibit age-dependent neuroaxonal dystrophy

  • Progressive motor abnormalities mimic human disease

  • TECPR2 antibodies facilitate tissue-level characterization of pathological changes

  • Helps validate therapeutic approaches targeting the affected pathways

These studies demonstrate that TECPR2 mutations disrupt multiple cellular pathways, with autophagy dysfunction being particularly prominent. The combined defects in autophagy, secretory transport, and endosomal recycling likely contribute to the progressive neurodegeneration observed in patients .

What are the methodological considerations for using TECPR2 antibodies in patient-derived samples?

Working with patient-derived samples presents unique challenges and opportunities for TECPR2 research. Key methodological considerations include:

• Sample-specific optimization:

  • Patient fibroblasts: Typically used as accessible primary cells

    • Optimize cell density and growth conditions for consistent TECPR2 expression

    • Consider passage number effects on autophagy phenotypes

    • Compare multiple control lines to account for individual variations

  • Induced pluripotent stem cells (iPSCs): Useful for generating neural lineages

    • Validate TECPR2 antibody performance in differentiated neural cells

    • Optimize fixation protocols for neural cultures

  • Post-mortem tissue: Challenging but informative

    • Account for post-mortem interval effects on protein preservation

    • Optimize antigen retrieval for formalin-fixed paraffin-embedded (FFPE) tissues

• Mutation-specific considerations:

  • Epitope accessibility: Ensure antibody epitope is preserved in patient mutations

    • For biotin-conjugated antibodies raised against region 516-783 AA, this epitope should be preserved in most patient mutations

  • Expression level variations: Some mutations may affect protein stability

    • Adjust exposure settings for low-expression samples

    • Consider signal amplification methods for detection of low abundance mutant proteins

• Experimental design:

  • Patient-control paired analysis: Process and analyze matched samples simultaneously

  • Blinded assessment: Perform quantification without knowledge of sample identity

  • Technical replicates: Include multiple technical replicates to control for processing variables

  • Age/sex matching: Control for demographic variables that may affect TECPR2 expression or function

• Fixation and processing considerations:

  • Sample preservation: Optimize protocols to maintain protein integrity

  • Permeabilization: Adjust for differences in membrane composition between patient and control cells

  • Antigen retrieval: May need optimization for specific mutations affecting protein conformation

• Validation approaches:

  • Complementary techniques: Combine antibody-based detection with mRNA analysis

  • Rescue experiments: Reintroduce wild-type TECPR2 into patient cells

  • Functional readouts: Correlate antibody staining with functional autophagy assays

  • Quantitative analysis: Develop robust quantification protocols for subtle phenotypic differences

Careful attention to these methodological considerations will maximize the value of patient-derived samples in TECPR2 research and ensure reliable, reproducible results that advance our understanding of disease mechanisms .

How might TECPR2 antibodies contribute to development of therapeutic approaches for SPG49/HSAN9?

TECPR2 antibodies can play crucial roles in developing therapeutic strategies for SPG49/HSAN9 through several research applications:

• Target validation and drug screening:

  • High-content screening assays using TECPR2 antibodies to monitor:

    • Restoration of proper TECPR2 localization

    • Recovery of autophagosome-lysosome fusion

    • Normalization of cargo recycling in endosomes

  • Validation of compounds that stabilize mutant TECPR2 or enhance remaining function

  • Identification of pathway modulators that bypass TECPR2 dysfunction

• Biomarker development:

  • Quantitative assessment of TECPR2 levels and localization in accessible patient samples

  • Correlation with disease progression and severity

  • Monitoring treatment response in clinical trials

  • Development of companion diagnostics for personalized medicine approaches

• Gene therapy development:

  • Validation of gene delivery efficiency using TECPR2 antibodies

  • Assessment of wild-type TECPR2 expression in transduced cells

  • Confirmation of proper subcellular localization and function

  • Correlation with phenotypic rescue in disease models

• Domain-specific therapeutic approaches:

  • Determination of minimal functional domains for therapeutic delivery

  • The C-terminal TECPR domain with intact LIR motif rescues autophagy in patient fibroblasts

  • TECPR2 antibodies can verify expression and localization of therapeutic constructs

  • Evaluation of domain-specific effects on different TECPR2 functions

• Disease mechanism stratification:

  • Different TECPR2 mutations may primarily affect:

    • Autophagy (LIR motif mutations)

    • Secretory pathway (mutations affecting ERES interaction)

    • Endosomal function (Rab5-binding domain mutations)

  • TECPR2 antibodies help categorize patients based on predominant mechanism

  • Enables personalized therapeutic approaches targeting specific pathways

These applications highlight how TECPR2 antibodies can contribute to translational research efforts aimed at developing effective therapies for these currently untreatable neurodegenerative conditions .

What emerging technologies might enhance the utility of TECPR2 antibodies in future research?

Several cutting-edge technologies show promise for expanding the applications of TECPR2 antibodies in neurodegenerative disease research:

• Advanced proximity labeling technologies:

  • TurboID and miniTurbo: Faster biotin ligases for temporal control of proximity labeling

  • Split-TurboID: For studying conditional protein-protein interactions involving TECPR2

  • Application to map dynamic TECPR2 interactomes in different subcellular compartments

  • Correlation with findings from conventional antibody-based approaches

• Super-resolution microscopy integration:

  • STORM/PALM: Nanoscale resolution of TECPR2 localization at autophagosome-lysosome contact sites

  • Expansion microscopy: Physical expansion of samples for improved resolution with standard confocal equipment

  • Correlative light and electron microscopy (CLEM): Combining antibody-based fluorescence with ultrastructural analysis

  • Lattice light-sheet microscopy: For dynamic studies of TECPR2 recruitment in live cells

• Single-cell analysis technologies:

  • Single-cell proteomics with TECPR2 antibodies

  • Mass cytometry (CyTOF) for multi-parameter analysis of TECPR2 pathways

  • Integration with single-cell transcriptomics for correlation studies

  • Spatial transcriptomics correlated with TECPR2 immunohistochemistry in brain tissues

• Artificial intelligence and image analysis:

  • Deep learning algorithms for automated quantification of TECPR2 localization patterns

  • Machine learning for identification of subtle phenotypic differences in patient samples

  • Predictive modeling of compound effects on TECPR2 function based on imaging data

  • Computer vision approaches for high-content screening applications

• Advanced in vitro and in vivo models:

  • Brain organoids with TECPR2 mutations for developmental studies

  • CRISPR-engineered animal models with specific TECPR2 domain mutations

  • Patient-derived neurons for personalized drug screening

  • TECPR2 antibodies essential for validation studies in these models

• Antibody engineering innovations:

  • Single-domain antibodies (nanobodies) against TECPR2 for improved penetration

  • Recombinant antibody fragments for intracellular expression to track TECPR2 in living cells

  • Bispecific antibodies targeting TECPR2 and interacting proteins simultaneously

  • Photoswitchable antibody conjugates for super-resolution applications

These emerging technologies, when combined with well-validated TECPR2 antibodies, will significantly advance our understanding of TECPR2 biology and its role in neurodegenerative disorders, potentially accelerating therapeutic development .