TIMM17B Antibody

What is TIMM17B and why is it important to study using antibodies?

TIMM17B (Translocase of Inner Mitochondrial Membrane 17 Homolog B) is a multipass transmembrane protein that forms an integral component of the mitochondrial translocase TIM23 complex. This complex plays a crucial role in facilitating the transport of transit peptide-containing proteins from the cytosol across the mitochondrial inner membrane into the mitochondrial matrix . TIMM17B is essential for mitochondrial function and cellular energy metabolism.

The protein has a molecular weight of approximately 18 kDa and consists of 172 amino acids . Studying TIMM17B using antibodies allows researchers to investigate its expression, localization, interactions, and functional role in normal physiology and disease states. Antibody-based detection methods provide valuable insights into mitochondrial protein import mechanisms, which are fundamental to understanding mitochondrial biogenesis and dysfunction in various pathological conditions.

What are the primary applications for TIMM17B antibodies in research?

TIMM17B antibodies are versatile tools employed in multiple experimental techniques, including:

These applications enable researchers to study TIMM17B expression patterns, protein-protein interactions, and functional roles in different experimental contexts .

What species reactivity can I expect from commercially available TIMM17B antibodies?

Based on the available research products, TIMM17B antibodies show different species reactivity profiles depending on the specific product:

When selecting an antibody, it's crucial to verify the specific species reactivity through validation data, as cross-reactivity can vary significantly between products. Many antibodies are raised against human TIMM17B but show cross-reactivity with mouse and rat due to high sequence homology across these species .

What is the optimal protocol for detecting TIMM17B via Western blotting?

For optimal Western blot detection of TIMM17B, the following protocol is recommended based on experimental validation data:

Sample Preparation:

• Prepare whole cell lysates or subcellular (mitochondrial) fractions using a buffer containing protease inhibitors.

• Determine protein concentration using BCA or Bradford assay.

• Load 20-40 μg of total protein per lane.

Electrophoresis and Transfer:

• Separate proteins on 12-15% SDS-PAGE (recommended due to TIMM17B's small size of 18 kDa).

• Transfer to PVDF or nitrocellulose membrane (PVDF often preferred for small proteins).

Detection:

• Block in 5% non-fat milk or BSA in TBST for 1 hour at room temperature.

• Incubate with primary TIMM17B antibody at 1:500-1:2000 dilution in blocking buffer overnight at 4°C .

• Wash 3-5 times with TBST.

• Incubate with HRP-conjugated secondary antibody (1:5000-1:10000) for 1 hour at room temperature.

• Wash 3-5 times with TBST.

• Develop using ECL substrate and visualize bands.

Expected Results:

• TIMM17B typically appears as a single band at 17-20 kDa .

• Positive controls include human heart tissue, liver tissue, and cell lines like PC-3, RT4, and U-251 MG, which express detectable levels of TIMM17B .

For troubleshooting non-specific bands, additional blocking or antibody titration may be required to optimize signal-to-noise ratio.

How should I optimize immunohistochemistry protocols for TIMM17B detection in tissue samples?

Successful IHC detection of TIMM17B in tissue samples requires careful optimization:

Tissue Preparation:

• Use formalin-fixed, paraffin-embedded (FFPE) tissue sections (4-6 μm thick).

• Deparaffinize completely in xylene and rehydrate through graded alcohols.

Antigen Retrieval (Critical Step):

• Heat-mediated antigen retrieval is essential for TIMM17B detection.

• Recommended methods based on validation data:

  • Citrate buffer (pH 6.0) for 15-20 minutes in a pressure cooker or microwave .

  • TE buffer (pH 9.0) as an alternative method that may yield better results for certain antibodies .

Staining Protocol:

• Block endogenous peroxidase activity with 3% H₂O₂ for 10 minutes.

• Block non-specific binding with 5-10% normal serum for 30 minutes.

• Incubate with primary TIMM17B antibody at 1:20-1:200 dilution overnight at 4°C or 1-2 hours at room temperature .

• Apply appropriate detection system (e.g., polymer-HRP secondary antibody).

• Develop with DAB substrate and counterstain with hematoxylin.

Validation Controls:

• Positive tissue controls: Human placenta, colon, and gastric tissues have demonstrated reliable TIMM17B expression .

• Negative controls: Omit primary antibody on duplicate sections.

Optimization may require testing multiple antibody dilutions and antigen retrieval conditions to determine the protocol that provides specific staining with minimal background.

What are the key considerations for designing experiments to study TIMM17B protein interactions?

Studying TIMM17B protein interactions requires careful experimental planning due to its location in the mitochondrial inner membrane and its role in the TIM23 complex:

Recommended Approaches:

• Co-Immunoprecipitation (Co-IP):

  • Use lysis buffers containing mild detergents (0.5-1% NP-40 or digitonin) to preserve protein complexes.

  • Immunoprecipitate with 0.5-4.0 μg TIMM17B antibody per 1-3 mg of protein lysate .

  • Validate results with reciprocal Co-IP using antibodies against suspected interaction partners.

• Proximity Ligation Assay (PLA):

  • Useful for detecting in situ protein interactions in fixed cells or tissues.

  • Requires antibodies raised in different species for TIMM17B and interaction partners.

• Bioluminescence Resonance Energy Transfer (BRET) or Fluorescence Resonance Energy Transfer (FRET):

  • For real-time interaction monitoring in living cells.

  • Requires genetic fusion of TIMM17B and partner proteins with appropriate reporter tags.

Key Considerations:

• Mitochondrial isolation may be necessary for enrichment of TIMM17B-containing complexes.

• Known interaction partners to consider include components of the TIM23 complex.

• Controls should include:

  • IgG isotype control for non-specific binding

  • Input samples (pre-IP lysate) for expression verification

  • Positive controls with established interaction partners

  • Negative controls with proteins known not to interact with TIMM17B

For membrane proteins like TIMM17B, crosslinking prior to lysis may help stabilize transient interactions, but optimize crosslinking conditions to avoid artifacts.

How can I validate TIMM17B antibody specificity to ensure reliable experimental results?

Rigorous validation of TIMM17B antibody specificity is crucial for generating trustworthy data. Multiple complementary approaches should be employed:

Validation Strategies:

• Genetic Knockout/Knockdown Controls:

  • Compare antibody signal in TIMM17B knockout/knockdown samples versus wildtype.

  • CRISPR/Cas9-mediated knockout or siRNA knockdown of TIMM17B provides the most definitive validation.

  • Expected result: Significant reduction or complete absence of signal in knockout/knockdown samples.

• Peptide Competition Assay:

  • Pre-incubate antibody with excess immunizing peptide before application to samples.

  • Expected result: Blocked antibody should show diminished or absent signal if specific.

• Multiple Antibody Validation:

  • Use multiple antibodies targeting different epitopes of TIMM17B.

  • Concordant results across antibodies increase confidence in specificity.

• Recombinant Expression:

  • Overexpress tagged TIMM17B in a cellular system and confirm antibody detection.

  • Verify molecular weight matches prediction (18 kDa plus tag size).

• Mass Spectrometry Confirmation:

  • Perform immunoprecipitation followed by mass spectrometry to confirm the identity of the pulled-down protein.

Documentation of Validation Results:

• Include validation controls in publications to demonstrate antibody specificity.

• Document lot-to-lot variation if observed, as antibody performance can vary between production batches.

Combining multiple validation approaches provides the strongest evidence for antibody specificity and increases confidence in experimental results.

What strategies can address challenges in detecting low-abundance TIMM17B in certain cell types or tissues?

Detecting low-abundance TIMM17B in certain experimental systems presents significant challenges that require specialized approaches:

Signal Enhancement Strategies:

• Sample Enrichment:

  • Isolate mitochondrial fractions to concentrate TIMM17B prior to analysis.

  • Use subcellular fractionation protocols optimized for mitochondrial proteins.

• Amplification Systems for IHC/ICC:

  • Employ tyramide signal amplification (TSA) to enhance chromogenic or fluorescent signals.

  • Use polymer-based detection systems that provide greater sensitivity than traditional secondary antibodies.

• Enhanced Western Blot Detection:

  • Use high-sensitivity ECL substrates specifically designed for low-abundance proteins.

  • Consider longer exposure times while monitoring background.

  • Load higher protein amounts (60-100 μg) if sample permits.

• Optimized Antibody Concentration:

  • Perform careful titration experiments to determine optimal antibody concentration.

  • For some systems, higher concentrations (1:100-1:200) may be necessary despite increased background risk .

Alternative Detection Methods:

• PCR-Based Detection:

  • RT-qPCR for TIMM17B mRNA as a complementary approach when protein detection is challenging.

  • Remember that mRNA and protein levels may not always correlate.

• Mass Spectrometry:

  • Consider targeted mass spectrometry approaches for very low abundance detection.

  • Requires specialized equipment and expertise but offers high specificity.

When publishing results from low-abundance systems, thorough documentation of methodology and appropriate controls is essential to demonstrate the validity of findings.

How can I investigate TIMM17B post-translational modifications using antibody-based approaches?

Investigating post-translational modifications (PTMs) of TIMM17B requires specialized antibodies and techniques:

Experimental Approaches:

Verification Strategies:

• Mutagenesis Validation:

  • Generate site-specific mutants of predicted PTM sites.

  • Compare antibody recognition between wild-type and mutant proteins.

• Physiological Relevance:

  • Investigate PTM changes in response to relevant physiological stimuli:

    • Mitochondrial stress conditions

    • Cell cycle phases

    • Different metabolic states

• PTM Crosstalk:

  • Consider how different modifications might interact or compete (e.g., phosphorylation affecting ubiquitination).

Currently, limited research has been published on specific PTMs of TIMM17B, making this an area with significant potential for novel discoveries.

How should I interpret conflicting results from different TIMM17B antibodies in the same experiment?

Conflicting results from different TIMM17B antibodies are not uncommon and require systematic investigation:

Analytical Approach:

• Epitope Mapping Analysis:

  • Determine the specific epitopes recognized by each antibody.

  • Antibodies targeting different domains of TIMM17B may show discrepant results if:

    • Certain epitopes are masked in protein complexes

    • Different isoforms or splice variants are present

    • Post-translational modifications affect epitope accessibility

• Correlation with Other Detection Methods:

  • Compare antibody results with mRNA expression (RT-qPCR).

  • If possible, correlate with mass spectrometry protein detection.

  • Consider functional assays to determine which antibody results align with expected biology.

• Systematic Validation:

  • Create a comparison table documenting the performance of each antibody:

• Technical Considerations:

  • Evaluate fixation/sample preparation effects on epitope accessibility.

  • Consider antibody format (polyclonal vs. monoclonal).

  • Assess lot-to-lot variation with the same antibody product.

Resolution Strategies:

• Define Clear Criteria for Positive Results:

  • Require concordance between at least two independent antibodies.

  • Prioritize antibodies with the strongest validation evidence.

• Report Discrepancies Transparently:

  • In publications, acknowledge conflicting results rather than selectively reporting.

  • Discuss possible biological or technical reasons for discrepancies.

• Functional Validation:

  • Use genetic approaches (knockdown/knockout) to determine which antibody most accurately reflects true protein expression.

What are the most common technical problems when using TIMM17B antibodies and how can they be resolved?

Researchers commonly encounter several technical challenges when working with TIMM17B antibodies:

Issue #1: Weak or Absent Signal in Western Blot

Issue #2: High Background in IHC/ICC

Issue #3: Immunoprecipitation Failures

Issue #4: Multiple Bands in Western Blot

Documenting all optimization steps and validations is essential for publication quality and reproducibility.

How do I accurately quantify TIMM17B expression levels across different experimental conditions?

Accurate quantification of TIMM17B expression requires careful attention to experimental design and analysis:

Quantification Best Practices:

• Western Blot Quantification:

  • Use internal loading controls appropriate for your experimental system:

    • Total protein normalization (Ponceau S, REVERT, etc.) is preferred over housekeeping proteins

    • If using housekeeping proteins, verify they're not affected by your experimental conditions

  • Ensure signal is within linear dynamic range:

    • Run a dilution series to confirm linearity of detection

    • Avoid saturated bands that underestimate differences

  • Use technical replicates (minimum 3) and biological replicates (minimum 3)

  • Employ image analysis software (ImageJ, Image Lab, etc.) for densitometry

  • Report normalized values (TIMM17B/loading control ratio)

• ELISA Quantification:

  • Generate standard curves with recombinant TIMM17B protein

  • Ensure samples fall within the linear range of the standard curve

  • Run samples in duplicate or triplicate

  • Include inter-assay controls to normalize between plates

• Immunohistochemistry Quantification:

  • Use computer-assisted image analysis for objective scoring

  • Define clear scoring criteria (H-score, percentage positive cells, intensity scales)

  • Blind the scorer to experimental conditions

  • Score multiple fields per sample (minimum 5)

  • Include positive and negative control tissues in each batch

Statistical Analysis:

• Appropriate Statistical Tests:

  • For comparing two groups: t-test (if normally distributed) or Mann-Whitney (if non-parametric)

  • For multiple groups: ANOVA with appropriate post-hoc tests

  • Report exact p-values rather than thresholds

• Data Presentation:

  • Include representative images of blots/staining

  • Present quantification as mean ± SEM or mean ± SD

  • Use appropriate graph types (bar graphs for categorical comparisons, scatter plots to show individual data points)

Control Experiments:

• Validation Controls:

  • Positive controls: Tissues/cells known to express TIMM17B (heart, liver, PC-3 cells)

  • Negative controls: TIMM17B-depleted samples (if available)

• Technical Controls:

  • For Western blot: Full blot images including molecular weight markers

  • For IHC: Serial sections with primary antibody omitted

When reporting changes in TIMM17B expression, consider both statistical significance and biological significance in your interpretation.

How can I use TIMM17B antibodies to investigate mitochondrial import defects in disease models?

TIMM17B antibodies can be powerful tools for investigating mitochondrial protein import defects in various disease contexts:

Experimental Design Approaches:

• Comparative Expression Analysis:

  • Quantify TIMM17B levels in affected versus control tissues/cells using validated antibodies.

  • Compare expression patterns across multiple disease states using tissue microarrays.

  • Correlate TIMM17B expression with mitochondrial function markers.

• Localization Studies:

  • Use immunofluorescence co-localization with mitochondrial markers (MitoTracker, TOMM20) to assess TIMM17B distribution.

  • Investigate potential mislocalization or aggregation in disease models.

  • Employ super-resolution microscopy for detailed subcellular distribution analysis.

• Functional Complex Assembly Analysis:

  • Use co-immunoprecipitation with TIMM17B antibodies to isolate TIM23 complex components.

  • Compare complex composition between healthy and disease samples.

  • Identify altered protein interactions that may explain import defects.

• Import Efficiency Assessment:

  • Combine with in vitro import assays using isolated mitochondria.

  • Use fluorescence-based reporters of mitochondrial import efficiency.

  • Correlate import defects with TIMM17B alterations.

Disease-Specific Applications:

Emerging Research Direction:
The relationship between TIMM17B dysfunction and mitochondrial stress responses presents a promising area for investigation. Researchers can employ stress markers (e.g., mitochondrial unfolded protein response factors) alongside TIMM17B detection to elucidate the consequences of import machinery defects on cellular homeostasis.

What are the latest methodological advances in TIMM17B research utilizing antibody-based approaches?

Recent methodological advances have expanded the utility of TIMM17B antibodies in cutting-edge research applications:

Emerging Technologies:

• Proximity Labeling Proteomics:

  • BioID or APEX2 fusion to TIMM17B to identify proximal proteins

  • Reveals dynamic interaction network of TIMM17B in living cells

  • Advantages: Captures weak or transient interactions missed by traditional co-IP

  • Applications: Mapping the complete interactome of TIMM17B at the mitochondrial inner membrane

• Super-Resolution Microscopy:

  • STORM/PALM imaging using fluorophore-conjugated TIMM17B antibodies

  • Achieves nanometer-scale resolution of TIMM17B distribution

  • Applications: Visualizing TIMM17B clusters and their relationship to import sites

• Single-Cell Protein Analysis:

  • Mass cytometry (CyTOF) incorporating metal-labeled TIMM17B antibodies

  • Single-cell Western blotting techniques

  • Applications: Heterogeneity analysis of TIMM17B expression in complex tissues

• Active Learning for Antibody Binding Prediction:

  • Recent research published in March 2025 describes novel active learning strategies for antibody-antigen binding prediction

  • Such approaches could improve TIMM17B antibody development and characterization

  • The best algorithms reduced the number of required antigen variants by up to 35%

Integrative Multi-Omics Approaches:

• Antibody-Based Proteomics Integrated with Transcriptomics:

  • Correlation of TIMM17B protein levels (detected by antibodies) with mRNA expression

  • Identifies post-transcriptional regulation mechanisms

  • Applications: Understanding regulatory networks controlling TIMM17B expression

• Spatial Transcriptomics with Protein Validation:

  • Combining spatial transcriptomics data with antibody-based protein localization

  • Provides tissue context for TIMM17B expression patterns

  • Applications: Mapping cell type-specific expression in heterogeneous tissues

Technological Challenges and Solutions:
Despite these advances, researchers face challenges with antibody specificity in complex applications. Cross-validation using orthogonal methods and rigorous antibody validation remain essential for reliable results with these sophisticated techniques.

How can I develop experimental designs to study TIMM17B's role in disease pathogenesis using antibody-based approaches?

Designing experiments to elucidate TIMM17B's role in disease pathogenesis requires multifaceted approaches combining antibody-based detection with functional analyses:

Comprehensive Experimental Framework:

• Expression Correlation Studies:

  • Design: Quantify TIMM17B levels across disease progression stages using validated antibodies.

  • Analysis: Correlate expression with:

    • Clinical parameters (disease severity, progression, patient survival)

    • Biochemical markers (mitochondrial function, oxidative stress)

    • Histopathological features

  • Expected Outcome: Identification of whether TIMM17B alterations precede or follow disease manifestation.

• Mechanistic Intervention Studies:

  • Design: Manipulate TIMM17B levels (overexpression/knockdown) in cell or animal disease models.

  • Analysis: Use antibodies to:

    • Confirm manipulation success

    • Track effects on TIM23 complex assembly

    • Monitor mitochondrial protein import efficiency

    • Assess cellular responses (apoptosis, stress responses)

  • Controls: Include rescue experiments to confirm specificity.

• Biomarker Development Pipeline:

  • Design: Evaluate TIMM17B as a potential disease biomarker using antibody-based detection.

  • Workflow:

    1. Initial discovery in small cohorts using Western blot

    2. Validation in larger cohorts using IHC on tissue microarrays

    3. Development of ELISA for potential clinical application

    4. Correlation with disease outcomes and treatment responses

  • Statistical Analysis: Determine sensitivity, specificity, and predictive value.

Disease-Specific Experimental Considerations:

Translational Research Directions:

• Therapeutic Target Assessment:

  • Use antibodies to validate TIMM17B as a potential therapeutic target.

  • Develop cell-based screening assays incorporating TIMM17B antibodies to identify compounds that modulate its function or expression.

• Precision Medicine Applications:

  • Develop IHC-based or ELISA-based methods to stratify patients based on TIMM17B status.

  • Correlate TIMM17B status with treatment responses.

When designing these studies, incorporate appropriate statistical power calculations, blinding procedures, and randomization to ensure robust and reproducible results.

What emerging research questions about TIMM17B could be addressed using antibody-based approaches?

Several cutting-edge research questions about TIMM17B remain unexplored and present exciting opportunities for antibody-based investigations:

Fundamental Biology Questions:

• Tissue-Specific Regulation:

  • How does TIMM17B expression vary across different tissues and cell types?

  • Are there tissue-specific binding partners that modify TIMM17B function?

  • Approach: Comprehensive tissue microarray analysis using validated antibodies to create an expression atlas.

• Developmental Dynamics:

  • How does TIMM17B expression change during embryonic development and cellular differentiation?

  • Does TIMM17B play a role in stem cell maintenance or differentiation?

  • Approach: Immunohistochemical analysis of developmental tissue series and differentiation models.

• Stress Response Mechanisms:

  • How is TIMM17B expression/localization altered under various cellular stresses?

  • Does TIMM17B play a role in mitochondrial stress signaling?

  • Approach: Combine antibody detection with stress pathway markers in stress response time courses.

Disease-Related Research Frontiers:

• Cancer Metabolism:

  • How does TIMM17B contribute to metabolic reprogramming in cancer cells?

  • Can TIMM17B expression serve as a prognostic biomarker in specific cancer types?

  • Approach: Correlative studies of TIMM17B levels with metabolic phenotypes across cancer subtypes.

• Neurodegenerative Diseases:

  • Does TIMM17B dysfunction contribute to protein aggregation in neurodegenerative disorders?

  • Are there disease-specific modifications of TIMM17B that affect its function?

  • Approach: Comparative analysis of TIMM17B in affected versus unaffected brain regions.

• Aging and Longevity:

  • How does TIMM17B expression/function change during aging?

  • Could modulation of TIMM17B activity influence lifespan or healthspan?

  • Approach: Age-dependent expression analysis in model organisms and human tissues.

Technological Innovation Opportunities:

• Single-Cell Proteomics:

  • Develop methods to detect TIMM17B at the single-cell level to understand cellular heterogeneity.

  • Approach: Adaptation of antibodies for mass cytometry or single-cell Western blot technologies.

• In Vivo Imaging:

  • Develop approaches to visualize TIMM17B dynamics in living systems.

  • Approach: Antibody fragment-based imaging probes for non-invasive detection.

These emerging questions represent areas where well-characterized TIMM17B antibodies could enable significant discoveries in mitochondrial biology and disease pathogenesis.

How can TIMM17B antibodies be integrated into high-throughput screening approaches for drug discovery?

TIMM17B antibodies can be integrated into various high-throughput screening (HTS) platforms to facilitate drug discovery targeting mitochondrial import pathways:

Antibody-Based HTS Assay Formats:

• Cell-Based Phenotypic Screening:

  • Assay Design: Use high-content imaging with fluorescently labeled TIMM17B antibodies to monitor:

    • Expression level changes

    • Subcellular localization shifts

    • Complex formation alterations

  • Advantages: Captures compound effects in intact cellular systems

  • Throughput: 10,000-100,000 compounds per screen

  • Detection: Automated microscopy with image analysis algorithms

• ELISA-Based Expression Screens:

  • Assay Design: Quantify TIMM17B protein levels in treated cells using sandwich ELISA

  • Application: Identify compounds that modulate TIMM17B expression

  • Throughput: >100,000 compounds per screen

  • Readout: Colorimetric or fluorescent detection

• Protein-Protein Interaction Disruption Screens:

  • Assay Format: AlphaScreen or TR-FRET using TIMM17B antibodies paired with antibodies against interaction partners

  • Target: Compounds that specifically modulate TIMM17B interactions with TIM23 complex components

  • Advantages: Cell-free system allows direct detection of interaction effects

  • Throughput: >500,000 compounds per screen

• Functional Import Assays with Antibody Validation:

  • Approach: Screen for compounds affecting mitochondrial protein import, then validate mechanism using TIMM17B antibodies

  • Workflow:

    1. Primary screen: reporter-based import efficiency assay

    2. Secondary screen: TIMM17B expression/localization using antibodies

    3. Mechanistic validation: effect on TIM23 complex assembly

Practical Implementation Considerations:

Data Analysis and Hit Selection:

• Quality Control Metrics:

  • Z'-factor >0.5 for assay robustness

  • Signal-to-background ratio >3

  • Coefficient of variation <15%

• Hit Selection Criteria:

  • Statistical significance (typically >3 standard deviations from control)

  • Dose-dependent effects

  • Lack of cytotoxicity at effective concentrations

  • Specificity for TIMM17B pathway versus general mitochondrial effects

• Machine Learning Integration:

  • Apply machine learning algorithms to high-content imaging data to identify subtle phenotypes

  • Use active learning strategies to improve antibody-antigen binding prediction for TIMM17B research