TIMM8B Antibody

What is the biological function of TIMM8B protein?

TIMM8B functions as a probable mitochondrial intermembrane chaperone that participates in the import and insertion of multi-pass transmembrane proteins into the mitochondrial inner membrane. It plays an essential role in the transfer of beta-barrel precursors from the TOM complex to the sorting and assembly machinery (SAM complex) of the outer membrane. The protein acts as a chaperone-like molecule that protects hydrophobic precursors from aggregation and guides them through the mitochondrial intermembrane space . This complex assists in importing and inserting essential proteins across mitochondrial membranes, which is crucial for mitochondrial respiratory chain function and cellular energy metabolism .

How do TIMM8B antibodies differ from antibodies against related proteins like TIMM8A?

While both TIMM8A and TIMM8B antibodies target proteins within the same family of mitochondrial translocases, they recognize distinct epitopes specific to each protein. TIMM8B antibodies are designed to bind specifically to the TIMM8B protein (also known as DDP2, DDPL, or TIM8B), while TIMM8A antibodies target the closely related TIMM8A protein .

The key difference lies in their research applications: TIMM8A has been extensively studied in relation to breast cancer (BRCA) and uterine corpus endometrial cancer (UCEC), with significant associations to prognosis and immune cell infiltration . TIMM8B research is less extensively documented in cancer biology, but the protein plays important roles in fundamental mitochondrial processes. When selecting an antibody, researchers must verify specificity through validation data to ensure the antibody recognizes only the intended target protein.

What sample types can be analyzed using TIMM8B antibodies?

TIMM8B antibodies can be used to analyze various biological sample types, including:

• Formalin-fixed paraffin-embedded (FFPE) tissue sections (IHC-P)

• Human tissue samples (particularly adrenal gland has been validated)

• Cell culture supernatants

• Plasma samples

• Serum samples

• Tissue homogenates

For optimal results, researchers should follow the manufacturer's recommended sample preparation protocols, including appropriate fixation methods for histological samples, proper dilution ranges, and validated incubation conditions .

How can TIMM8B expression be correlated with mitochondrial dysfunction in disease models?

Investigating TIMM8B expression in relation to mitochondrial dysfunction requires a multi-parametric approach:

• Quantitative assessment: Use TIMM8B antibodies in Western blotting or ELISA to quantify expression levels in disease models compared to controls .

• Spatial analysis: Employ immunohistochemistry with TIMM8B antibodies to examine localization patterns within tissue sections, focusing on regions with known mitochondrial abnormalities .

• Functional correlation: Pair TIMM8B expression data with mitochondrial function assays, including:

  • Oxygen consumption rate measurements

  • Membrane potential assessments

  • ATP production quantification

  • Reactive oxygen species detection

• Co-localization studies: Combine TIMM8B antibody staining with markers of mitochondrial stress (e.g., PINK1, Parkin) to evaluate the relationship between TIMM8B expression and mitophagy processes .

This integrated approach can reveal whether alterations in TIMM8B expression precede, coincide with, or follow mitochondrial dysfunction in specific disease contexts.

What are the implications of TIMM8B in immune cell function based on what we know about related mitochondrial proteins?

While direct evidence specifically on TIMM8B's role in immune function is limited in the provided search results, we can draw insights from research on the related protein TIMM8A:

• Potential immune regulatory mechanisms: TIMM8A expression shows significant correlation with immune cell infiltration in breast and uterine cancers, particularly with:

  • Th2 CD4+ T cells (strong positive correlation)

  • CD8+ T cells (positive correlation in BRCA but negative in UCEC)

  • Macrophages (variable correlation patterns)

  • Dendritic cells and neutrophils

• Mitochondrial integrity connection: As TIMM8B functions in maintaining mitochondrial protein import, dysregulation might impact immune cell metabolism, which is critical for proper immune function. Mitochondrial dynamics significantly influence T cell activation, macrophage polarization, and antigen presentation .

• Potential research direction: Investigators could examine TIMM8B expression in isolated immune cell populations and correlate with:

  • Cell activation status

  • Metabolic profiles (glycolysis vs. oxidative phosphorylation)

  • Cytokine production

  • Migration and effector functions

This approach would help determine if TIMM8B, like TIMM8A, plays a role in modulating immune responses through mitochondrial mechanisms.

How might TIMM8B antibody-based research contribute to understanding mitophagy in cancer progression?

TIMM8B antibody-based research could provide valuable insights into mitophagy mechanisms in cancer through several investigative approaches:

• Expression correlation studies: Using TIMM8B antibodies to quantify protein levels across cancer stages to establish patterns similar to those observed with TIMM8A, which shows increased expression with advancing cancer stages in BRCA and UCEC .

• Mitophagy marker co-localization: Combining TIMM8B immunostaining with mitophagy markers (LC3, PINK1, Parkin) to visualize spatial relationships during cancer progression.

• Functional mitochondrial assessment: Correlating TIMM8B expression with:

  • Mitochondrial mass (using MitoTracker staining)

  • Membrane potential (TMRM or JC-1 dyes)

  • Mitochondrial morphology (electron microscopy)

  • ATP production capacity

• Immune microenvironment analysis: Investigating relationships between TIMM8B expression and immune cell infiltration patterns, particularly focusing on:

  • CD8+ T cells and NK cells (cytotoxic responses)

  • Myeloid-derived suppressor cells (MDSCs)

  • Tumor-associated M2 macrophages (TAM M2)

This multifaceted approach could reveal whether TIMM8B influences cancer progression through mitochondrial quality control mechanisms and subsequent effects on tumor metabolism and immune evasion.

What are the optimal protocols for using TIMM8B antibodies in immunohistochemistry?

Recommended Protocol for TIMM8B Immunohistochemistry on FFPE Tissues:

• Sample preparation:

  • Fix tissues in 10% neutral buffered formalin

  • Process and embed in paraffin

  • Section at 4-6 μm thickness

  • Mount on positively charged slides

• Deparaffinization and rehydration:

  • Xylene: 2 changes, 5 minutes each

  • 100% ethanol: 2 changes, 3 minutes each

  • 95%, 80%, 70% ethanol: 3 minutes each

  • Distilled water: 5 minutes

• Antigen retrieval:

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0)

  • Pressure cook or microwave for 10-20 minutes

  • Cool to room temperature (approximately 20 minutes)

• Blocking and antibody application:

  • Block endogenous peroxidase: 3% H₂O₂ for 10 minutes

  • Protein block: 5% normal goat serum for 30 minutes

  • Primary antibody application: Dilute TIMM8B antibody to 1/100 ratio

  • Incubate at 4°C overnight or at room temperature for 1 hour

  • Wash in PBS or TBS: 3 times, 5 minutes each

• Detection and visualization:

  • Apply appropriate secondary antibody (typically HRP-conjugated)

  • Develop with DAB substrate

  • Counterstain with hematoxylin

  • Dehydrate, clear, and mount

• Controls:

  • Positive control: Human adrenal gland tissue (validated)

  • Negative control: Omit primary antibody

  • Isotype control: Use non-specific IgG at the same concentration

Human adrenal gland tissue has been validated for TIMM8B antibody staining and can serve as an appropriate positive control .

What approaches should be used to validate TIMM8B antibody specificity?

Comprehensive validation of TIMM8B antibody specificity requires multiple complementary approaches:

• Western blot analysis:

  • Run samples with known TIMM8B expression

  • Verify single band at expected molecular weight

  • Compare with recombinant TIMM8B protein standard

  • Include negative control samples

• Knockdown/knockout validation:

  • Test antibody on TIMM8B-depleted samples (siRNA, CRISPR)

  • Confirm signal reduction/elimination in depleted samples

  • Include scrambled/control treatments for comparison

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide

  • Apply to duplicate samples in parallel with untreated antibody

  • Verify signal reduction with peptide-blocked antibody

• Cross-reactivity assessment:

  • Test against recombinant TIMM8A and other related proteins

  • Evaluate staining patterns in tissues with differential expression

  • Confirm specificity across species if using for comparative studies

• Multiple antibody comparison:

  • Test different antibodies targeting distinct TIMM8B epitopes

  • Compare staining patterns and quantification results

  • Evaluate concordance between antibodies

This validation pipeline ensures that experimental findings truly reflect TIMM8B biology rather than non-specific or off-target antibody interactions.

How can TIMM8B ELISA assays be optimized for different sample types?

Optimization Strategies for TIMM8B ELISA Across Sample Types:

General Optimization Steps:

• Sample preparation standardization:

  • Establish consistent collection and processing protocols

  • Use appropriate protease inhibitors for all sample types

  • Standardize freeze-thaw cycles (limit to ≤2)

• Assay condition optimization:

  • Incubation temperature: Test 4°C, room temperature, and 37°C

  • Incubation time: Evaluate 1-hour, 2-hour, and overnight options

  • Washing steps: Compare manual vs. automated washing efficiency

• Signal enhancement strategies:

  • Evaluate signal amplification systems

  • Optimize substrate development time

  • Determine optimal enzyme conjugate dilution

• Quality control measures:

  • Include spike-recovery tests to assess matrix effects

  • Run parallelism tests for linearity verification

  • Establish intra- and inter-assay variation limits

These optimization approaches can help researchers achieve consistent, sensitive detection of TIMM8B across different experimental systems and sample types .

How do findings on TIMM8A in cancer biology inform potential research directions for TIMM8B?

The established research on TIMM8A provides a valuable framework for investigating TIMM8B's potential roles in cancer:

• Prognostic value assessment: TIMM8A has demonstrated significant associations with poor prognosis in breast cancer (BRCA) and uterine corpus endometrial cancer (UCEC) . Researchers should examine whether TIMM8B expression similarly correlates with patient outcomes across multiple cancer types.

• Cancer stage progression analysis: TIMM8A expression increases with advancing cancer stages . TIMM8B should be evaluated across cancer progression series using antibody-based tissue microarray analysis to determine if it shows similar patterns.

• Immune infiltration correlations: TIMM8A shows significant correlations with immune cell infiltration, particularly:

  • Positive correlation with Th2 CD4+ T cells in both BRCA and UCEC

  • Differential correlation with CD8+ T cells (positive in BRCA, negative in UCEC)

  • Variable associations with other immune cell populations

  Researchers should investigate whether TIMM8B shows similar immune correlation patterns or distinct immunological associations.

• Mitophagy pathway investigations: TIMM8A appears to influence immune infiltration and prognosis in cancer by affecting mitophagy . Given TIMM8B's role in mitochondrial protein import, researchers should examine its potential impact on mitochondrial quality control mechanisms in cancer cells.

This comparative approach leverages existing knowledge about TIMM8A to guide hypothesis-driven research on TIMM8B, potentially revealing whether these related proteins have overlapping or distinct roles in cancer biology.

What techniques can be employed to study the interaction between TIMM8B and other components of the mitochondrial import machinery?

Comprehensive Techniques for Studying TIMM8B Protein Interactions:

• Co-immunoprecipitation (Co-IP):

  • Use TIMM8B antibodies to pull down protein complexes

  • Identify interacting partners via mass spectrometry

  • Confirm specific interactions with Western blotting

  • Compare interaction profiles under different cellular conditions

• Proximity-based labeling:

  • Generate TIMM8B fusion with BioID or APEX2

  • Identify proximal proteins through biotinylation

  • Map the spatial organization of the TIMM8B interactome

  • Distinguish stable from transient interactions

• Fluorescence resonance energy transfer (FRET):

  • Tag TIMM8B and potential partners with appropriate fluorophores

  • Measure energy transfer indicating molecular proximity

  • Perform live-cell imaging to capture dynamic interactions

  • Quantify interaction strength through FRET efficiency calculations

• Crosslinking mass spectrometry (XL-MS):

  • Stabilize protein complexes with chemical crosslinkers

  • Identify crosslinked peptides through specialized MS workflows

  • Generate distance constraints for structural modeling

  • Map interaction interfaces at amino acid resolution

• Yeast two-hybrid screening:

  • Use TIMM8B as bait to screen for mitochondrial protein interactions

  • Validate positive hits with orthogonal methods

  • Map interaction domains through truncation mutants

  • Assess conservation of interactions across species

These methodologies, particularly when used in combination, can provide multi-layered evidence for TIMM8B's functional interactions within the mitochondrial protein import machinery, offering insights into both structural organization and functional relationships.

How might TIMM8B antibodies be utilized in studying neurodegenerative diseases with mitochondrial involvement?

TIMM8B antibodies could serve as valuable tools for investigating neurodegenerative disease mechanisms through several targeted approaches:

• Expression pattern analysis in disease models:

  • Quantify TIMM8B protein levels in brain tissues from neurodegenerative disease models

  • Compare expression patterns across different brain regions (cortex, hippocampus, substantia nigra)

  • Correlate TIMM8B expression with disease progression markers

  • Examine cell-type specific expression using co-localization with neuronal, glial, and microglial markers

• Mitochondrial dynamics assessment:

  • Evaluate TIMM8B localization during mitochondrial fragmentation/fusion events

  • Correlate TIMM8B expression with mitochondrial morphology changes

  • Assess relationship between TIMM8B levels and mitophagy markers in neurons

  • Determine if TIMM8B expression changes precede visible mitochondrial defects

• Intervention studies:

  • Monitor TIMM8B expression changes following treatments targeting mitochondrial function

  • Assess whether TIMM8B levels correlate with therapeutic outcomes

  • Evaluate TIMM8B as a potential biomarker for treatment response

• Patient sample analysis:

  • Compare TIMM8B levels in accessible patient samples (CSF, blood) with disease severity

  • Examine post-mortem brain tissue for alterations in TIMM8B expression pattern

  • Correlate findings with known genetic risk factors for mitochondrial dysfunction

Since mitochondrial dysfunction is implicated in conditions like Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis, TIMM8B antibodies could help elucidate disease-specific alterations in mitochondrial protein import machinery that might contribute to neurodegeneration.

What experimental designs would best elucidate the functional significance of TIMM8B in cellular stress responses?

Comprehensive Experimental Framework for TIMM8B in Stress Responses:

• Stress induction models with TIMM8B monitoring:

  • Oxidative stress: H₂O₂, paraquat, rotenone treatment

  • ER stress: tunicamycin, thapsigargin exposure

  • Hypoxia: controlled O₂ reduction

  • Nutrient deprivation: glucose/amino acid restriction

  • Monitor TIMM8B expression, localization, and post-translational modifications

• Loss-of-function approaches:

  • CRISPR/Cas9 knockout of TIMM8B

  • siRNA-mediated knockdown with titrated efficiency

  • Expression of dominant-negative TIMM8B variants

  • Assess impact on:

    • Mitochondrial membrane potential

    • ROS production

    • ATP synthesis capacity

    • Cell survival under stress conditions

• Rescue experiments:

  • Reintroduce wild-type or mutant TIMM8B into knockout cells

  • Evaluate domain-specific contributions to stress response

  • Assess compensatory mechanisms (upregulation of other TIM proteins)

  • Determine structure-function relationships

• Temporal analysis:

  • Time-course experiments during stress application and recovery

  • Live-cell imaging with tagged TIMM8B to track dynamics

  • Correlation with mitochondrial morphology changes

  • Identification of critical time points for intervention

• Multi-omics integration:

  • Transcriptomics: mRNA changes in response to TIMM8B manipulation

  • Proteomics: alterations in mitochondrial protein composition

  • Metabolomics: shifts in metabolic pathways

  • Network analysis to identify key TIMM8B-dependent processes

This systematic approach would provide comprehensive insights into TIMM8B's functional significance during cellular stress, potentially revealing therapeutic targets for conditions involving mitochondrial dysfunction.