TIMM9 Antibody

What is TIMM9 and why is it important in research?

TIMM9 is a molecular chaperone located in the mitochondrial intermembrane space (IMS) that plays an essential role in the transport of proteins destined for the mitochondrial inner membrane . It functions as part of a heterohexamer complex with TIMM10, forming an arrangement of 3 TIMM9 and 3 TIMM10 molecules . This complex mediates the substrate specificity of the TIM22 mitochondrial import pathway.

Recent research has identified TIMM9 as significantly associated with:

• Tumorigenesis

• Pathological stage progression

• Metastasis in multiple cancers

TIMM9 is particularly important for researchers investigating:

• Mitochondrial protein import mechanisms

• Cancer biomarkers and prognostic indicators

• Metabolic alterations in cancer cells

• Cell cycle progression

• Oxidative phosphorylation

• TCA cycle activity

What applications are TIMM9 antibodies suitable for?

TIMM9 antibodies have been validated for multiple applications, with specific optimization parameters depending on the antibody clone. Based on current commercial offerings, these antibodies are suitable for:

For optimal results, researchers should perform titration experiments to determine the ideal concentration for their specific sample type and experimental conditions .

How does the sensitivity of TIMM9 ELISA compare to other detection methods?

The TIMM9 ELISA kits available commercially offer excellent sensitivity with detection ranges typically from 46.88-3000 pg/mL and minimum detectable doses (MDD) of approximately 23.4 pg/mL . This sensitivity is determined by adding two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration.

For quantitative analysis:

• ELISA provides the most precise quantification with intra-assay CV<10% and inter-assay CV<15%

• Western blotting is semi-quantitative and useful for relative expression comparisons

• IHC/ICC provides spatial information but is less quantitative

Recovery rates in different matrices:

• Cell Culture Media: Average 100% (range 80-115%)

• Serum: Average 86% (range 84-107%)

What is the specificity of commonly available TIMM9 antibodies?

TIMM9 antibodies vary in their specificity depending on the immunogen used and purification methods. Most commercial antibodies:

• Target specific epitopes, often in the C-terminal region

• Detect endogenous levels of total TIMM9 protein

• Show minimal cross-reactivity with other proteins

For example, ABIN6265577 antibody specificity details:

• Targets the C-terminal region of human TIMM9

• Is purified by peptide affinity chromatography using SulfoLink TMCoupling Resin

• Has been validated to detect endogenous levels of total TIMM9

For validation, researchers often confirm specificity through:

• Western blot analysis showing a single band at the expected molecular weight (~10 kDa)

• Positive and negative control tissues

• Knockdown or knockout validation

How can TIMM9 antibodies be optimized for cancer tissue microarray analysis?

When optimizing TIMM9 antibodies for cancer tissue microarray (TMA) analysis, researchers should consider:

Preanalytical Variables:

• Fixation method and duration (typically 4-24 hours in 10% neutral buffered formalin)

• Antigen retrieval method (heat-induced epitope retrieval with citrate buffer pH 6.0 or EDTA buffer pH 9.0)

• Blocking protocol (3-5% BSA or normal serum from the same species as secondary antibody)

Analytical Variables:

• Primary antibody dilution (start with manufacturer's recommendation and titrate)

• Incubation time and temperature (overnight at 4°C often yields best signal-to-noise ratio)

• Detection system (polymer-based systems often provide superior sensitivity)

Post-analytical Considerations:

• Scoring system design (H-score, Allred score, or percentage of positive cells)

• Digital image analysis parameters

• Statistical analysis methodology

For prognostic studies, TIMM9 expression has been shown to correlate with patient survival in multiple cancer types . When designing TMAs, ensure sufficient representation of different tumor grades, stages, and normal adjacent tissue controls.

What are the optimal protocols for co-immunoprecipitation of TIMM9 with its interacting partners?

For successful co-immunoprecipitation (Co-IP) of TIMM9 with its interacting partners (particularly TIMM10 and other components of the mitochondrial import machinery), consider this optimized protocol:

Cell Lysis Buffer Composition:

• 20 mM Tris-HCl (pH 7.5)

• 150 mM NaCl

• 1 mM EDTA

• 1% Triton X-100 or 1% Digitonin (preserves membrane protein interactions better)

• 10% glycerol

• Protease inhibitor cocktail

• Phosphatase inhibitors (if studying phosphorylation)

Step-by-Step Protocol:

• Prepare mitochondrial fraction using differential centrifugation or commercial isolation kit

• Lyse mitochondria in buffer (mild conditions to preserve protein-protein interactions)

• Pre-clear lysate with Protein A/G beads (1 hour at 4°C)

• Incubate pre-cleared lysate with TIMM9 antibody (5 μg per 1 mg protein) overnight at 4°C

• Add Protein A/G beads and incubate for 2-4 hours at 4°C

• Wash 4-5 times with washing buffer (lysis buffer with reduced detergent)

• Elute with sample buffer or gentle elution buffer

• Analyze by western blotting

Important Controls:

• IgG isotype control

• Input sample (5-10% of lysate used for IP)

• Reverse IP (using antibody against suspected interacting partner)

The most reliable interacting partner identified through molecular simulations is ITFG1, which shows promising structural complementarity with TIMM9 . The TIMM9-TIMM10 heterohexamer complex is also well-established and can serve as a positive control.

How can TIMM9 antibodies be used to investigate mitochondrial dysfunction in cancer?

TIMM9 antibodies can be powerful tools for investigating mitochondrial dysfunction in cancer through several methodological approaches:

Immunohistochemistry Profiling:

• Compare TIMM9 expression across cancer types and correlate with clinical outcomes

• Use dual staining with other mitochondrial markers (TOMM20, COX4) to assess mitochondrial integrity

• Analyze subcellular localization patterns in different tumor stages

Functional Analysis:

• Isolate mitochondria from cancer cell lines with varying TIMM9 expression levels

• Assess import efficiency of reporter proteins using in vitro import assays

• Correlate TIMM9 expression with:

  • Oxygen consumption rate (OCR)

  • Extracellular acidification rate (ECAR)

  • ATP production

  • Reactive oxygen species (ROS) levels

Integration with Multi-Omics Data:
Research has revealed that TIMM9 is positively related to:

• Cell cycle progression

• Mitochondrial and ribosomal function

• Oxidative phosphorylation

• TCA cycle activity

• Innate and adaptive immunity

These associations can be further investigated using TIMM9 antibodies in combination with other molecular techniques to build a comprehensive picture of mitochondrial dysfunction mechanisms in cancer.

What are the considerations when using TIMM9 antibodies for detecting mutations and variants?

When using antibodies to detect TIMM9 mutations or variants, especially the S49L variant associated with poor cancer prognosis , researchers should consider:

Epitope Mapping:

• Determine if the antibody epitope overlaps with the mutation site

• For S49L detection, ensure the antibody recognizes the region containing amino acid 49

• Consider using multiple antibodies targeting different epitopes

Mutation-Specific Antibody Approaches:

• Use antibodies specifically raised against the mutant peptide sequence

• Perform competition assays with wild-type and mutant peptides

• Validate specificity using cells expressing wild-type vs. mutant TIMM9

Alternative Detection Methods:

• Combine antibody-based detection with genetic analysis

• Use proximity ligation assay (PLA) to detect specific protein-protein interactions affected by mutations

• Consider mass spectrometry-based approaches for definitive variant identification

Controls and Validation:

• Include known positive samples for the variant of interest

• Use CRISPR/Cas9-engineered cell lines expressing specific TIMM9 variants

• Validate findings using orthogonal methods (e.g., sequencing)

The S49L variant specifically has been associated with poor prognosis in cancer patients, making its accurate detection particularly relevant for translational research .

How can TIMM9 antibodies be used in combination with other techniques to study cancer metabolism?

Integrating TIMM9 antibody-based methods with other techniques provides powerful insights into cancer metabolism:

Metabolic Flux Analysis + Immunoprecipitation:

• Perform stable isotope labeling (13C-glucose, 13C-glutamine)

• Immunoprecipitate TIMM9 complexes from labeled cells

• Analyze associated proteins and metabolites using mass spectrometry

• Correlate findings with metabolic pathway activities

Spatial Metabolomics + Immunofluorescence:

• Co-localize TIMM9 with metabolic intermediates using IF and imaging mass spectrometry

• Map spatial distribution of TIMM9 in relation to hypoxic regions (using HIF1α staining)

• Correlate TIMM9 expression with metabolic gradients in tumor sections

Regulatory Network Analysis:
Recent research shows TIMM9 can be regulated by cancer-associated signaling pathways, such as the mTOR pathway . This connection can be investigated by:

• Treating cells with mTOR inhibitors (rapamycin, torin)

• Monitoring changes in TIMM9 expression/localization using antibodies

• Assessing downstream metabolic effects

This integrated approach allows researchers to establish causal relationships between TIMM9 expression and metabolic reprogramming in cancer cells.

How can I troubleshoot weak or absent signal when using TIMM9 antibodies in immunoblotting?

When encountering weak or absent signals in TIMM9 immunoblotting, consider the following systematic troubleshooting approach:

Sample Preparation Issues:

• TIMM9 is a small protein (~10 kDa) - use appropriate gel percentage (15-20%)

• Ensure complete denaturation (heat samples at 95°C for 5 minutes)

• Add protease inhibitors during lysis to prevent degradation

• Consider enriching mitochondrial fraction to increase signal

Transfer Problems:

• Use PVDF membrane (0.2 μm pore size) for small proteins

• Consider semi-dry transfer with specialized buffers for small proteins

• Reduce transfer time or voltage to prevent small proteins from passing through membrane

Detection Optimization:

• Increase primary antibody concentration (try 1:250 - 1:500)

• Extend primary antibody incubation (overnight at 4°C)

• Use a more sensitive detection system (ECL Prime or Femto)

• Optimize blocking conditions (try 5% BSA instead of milk for phospho-specific antibodies)

Control Experiments:

• Include positive control (tissue/cell line known to express TIMM9)

• Verify protein loading with housekeeping proteins

• Test multiple TIMM9 antibodies targeting different epitopes

Testing a panel of cell lines with known TIMM9 expression levels can help establish the detection sensitivity of your antibody and protocol.

What are the best practices for quantifying TIMM9 expression in tissue samples?

For reliable quantification of TIMM9 expression in tissue samples, consider these best practices:

Immunohistochemistry Quantification:

• Use digital pathology software for objective measurement

• Develop a scoring system accounting for both staining intensity and percentage of positive cells

• Consider H-score method: ∑(i × Pi) where i = intensity (0-3) and Pi = percentage of positive cells

• Include internal controls in each batch for normalization

Western Blot Quantification:

• Use housekeeping proteins appropriate for your tissue type

• Consider stain-free technology for total protein normalization

• Ensure you're in the linear dynamic range of detection

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

ELISA-Based Quantification:
TIMM9 ELISA kits offer quantitative detection with:

• Detection range: 46.88-3000 pg/mL

• Minimum detectable dose: ~23.4 pg/mL

• Intra-plate precision: CV<10%

• Inter-plate precision: CV<15%

For tissue homogenates, the linearity of dilution ranges from 80-105% across different dilution factors (1:2, 1:4, 1:8, 1:16) , indicating reliable quantification across a range of concentrations.

How should I validate TIMM9 antibody specificity for my experimental system?

Thorough validation of TIMM9 antibody specificity is essential for reliable research findings. Consider this comprehensive validation strategy:

Genetic Approaches:

• siRNA/shRNA knockdown of TIMM9 followed by western blot

• CRISPR/Cas9 knockout of TIMM9 as negative control

• Overexpression of tagged TIMM9 as positive control

Biochemical Validation:

• Peptide competition assay with immunizing peptide

• Test multiple antibodies targeting different epitopes

• Immunoprecipitation followed by mass spectrometry

Control Samples:

• Test tissues/cells known to express high vs. low levels of TIMM9

• Include species controls to confirm cross-reactivity claims

• Analyze subcellular localization (should be primarily mitochondrial)

Application-Specific Validation:
For IHC/ICC:

• Include appropriate blocking controls

• Test on known positive and negative tissues

• Perform subcellular localization studies to confirm mitochondrial pattern

For WB:

• Confirm single band at expected molecular weight (~10 kDa)

• Verify signal reduction upon TIMM9 knockdown

• Test antibody performance in different lysis buffers

How can TIMM9 antibodies be used to investigate the role of mitochondrial dysfunction in neurodegenerative diseases?

While TIMM9 has been extensively studied in cancer, its role in neurodegenerative diseases is an emerging area of research. TIMM9 antibodies can be valuable tools in this field:

Tissue-Based Studies:

• Compare TIMM9 expression and localization in post-mortem brain tissues from patients with neurodegenerative diseases versus healthy controls

• Assess co-localization with markers of mitochondrial dysfunction or proteinopathy

• Evaluate regional variation in expression across brain structures affected in different neurodegenerative conditions

Cellular Models:

• Use patient-derived iPSCs differentiated into neurons to study TIMM9 dynamics

• Apply TIMM9 antibodies to monitor mitochondrial import efficiency in cellular models of neurodegeneration

• Investigate interactions between TIMM9 and disease-associated proteins

Mechanistic Investigations:
TIMM9 is interconnected with disorders involving TIMM10 since they jointly facilitate mitochondrial function, indicating that disruptions in either protein can lead to related pathophysiological conditions where energy-metabolizing tissues like those in the brain and muscles are most affected .

Research protocols should include:

• Assessment of mitochondrial morphology and distribution

• Measurement of mitochondrial membrane potential

• Analysis of protein import efficiency

• Evaluation of ROS production and oxidative stress markers

Combining TIMM9 antibody-based techniques with functional measures of mitochondrial health can provide insights into the role of mitochondrial protein import in neurodegenerative processes.

What is the significance of TIMM9 as a prognostic biomarker in cancer research?

Recent research has established TIMM9 as a significant prognostic biomarker across multiple cancer types:

Research Findings:

• Overexpression of TIMM9 is significantly associated with tumorigenesis, pathological stage progression, and metastasis

• Missense mutations (particularly the S49L variant) and copy number variations in TIMM9 correlate with poor cancer prognosis

• TIMM9 is positively related to cell cycle progression, mitochondrial function, oxidative phosphorylation, and TCA cycle activity

Methodological Approaches for Prognostic Studies:

• Perform survival analysis based on TIMM9 expression levels

• Use multivariate Cox regression to adjust for confounding factors

• Develop risk prediction models incorporating TIMM9 expression

• Validate findings across independent patient cohorts

Technical Considerations for Biomarker Development:

• Standardize antibody-based detection methods for clinical application

• Establish expression cutoff values for prognostic stratification

• Combine with other biomarkers for improved predictive accuracy

• Consider both protein expression and genetic alterations

The implementation of TIMM9 as a prognostic biomarker requires careful standardization of immunohistochemical protocols and scoring systems to ensure reproducibility across laboratories and clinical settings.

How can researchers use TIMM9 antibodies to explore the relationship between mitochondrial function and cancer metabolism?

TIMM9 antibodies can be instrumental in elucidating the complex relationship between mitochondrial function and cancer metabolism:

Research Strategies:

• Metabolic Phenotyping:

  • Correlate TIMM9 expression levels with metabolic profiles of cancer cells

  • Compare oxidative phosphorylation vs. glycolytic capacity in cells with varying TIMM9 expression

  • Investigate the impact of TIMM9 modulation on metabolic flexibility

• Protein Import Analysis:

  • Use TIMM9 antibodies to assess the efficiency of mitochondrial protein import in cancer cells

  • Investigate how alterations in import machinery affect mitochondrial function and metabolic pathways

  • Correlate import efficiency with metabolic phenotypes

• Stress Response Studies:

  • Examine how TIMM9 expression changes under metabolic stress conditions

  • Investigate the role of TIMM9 in mediating adaptation to nutrient deprivation or hypoxia

  • Assess the impact of TIMM9 on cancer cell survival under metabolic stress

Experimental Approaches:

• Immunoprecipitation of TIMM9 complexes followed by proteomic analysis

• ChIP-seq to identify transcription factors regulating TIMM9 expression

• Live cell imaging with tagged TIMM9 to monitor dynamics during metabolic shifts

Research has revealed that TIMM9 could be regulated by cancer-associated signaling pathways, such as the mTOR pathway , providing a direct link between oncogenic signaling and mitochondrial function that warrants further investigation.

How can I develop a multiplex immunofluorescence protocol using TIMM9 antibodies for spatial analysis of tumor microenvironments?

Developing a multiplex immunofluorescence protocol incorporating TIMM9 antibodies requires careful optimization:

Protocol Development:

• Antibody Panel Design:

  • Select antibodies with non-overlapping species or isotypes

  • Consider the following panel for tumor microenvironment analysis:

    • TIMM9 (rabbit polyclonal)

    • Mitochondrial marker (mouse anti-TOMM20)

    • Cell type-specific markers (CD68 for macrophages, CD3 for T cells)

    • Metabolic markers (MCT1, GLUT1)

    • Proliferation marker (Ki67)

• Sequential Staining Approach:

  • Start with TIMM9 antibody at optimal dilution (typically 1:50 - 1:100 for IF)

  • Use tyramide signal amplification (TSA) for signal enhancement

  • Strip antibodies using heat or chemical methods between rounds

  • Proceed with subsequent antibodies in order of robustness

• Image Acquisition and Analysis:

  • Use multispectral imaging systems for spectral unmixing

  • Employ automated cell segmentation algorithms

  • Perform spatial analysis to identify cell-cell interactions and niches

  • Quantify TIMM9 expression in relation to other markers and spatial location

Quality Control Measures:

• Single-color controls for spectral unmixing

• Fluorescence minus one (FMO) controls

• Cross-reactivity testing between antibodies

• Batch correction methods for multi-slide analysis

This approach allows researchers to investigate the heterogeneity of TIMM9 expression within tumor microenvironments and correlate with functional and metabolic characteristics of different cell populations.

What are the considerations for using TIMM9 antibodies in flow cytometry applications?

Using TIMM9 antibodies for flow cytometry requires special considerations due to its mitochondrial localization:

Sample Preparation:

• Cell Permeabilization Options:

  • For whole cell analysis: Fix with 4% PFA followed by permeabilization with 0.1% Triton X-100

  • For isolated mitochondria: Gentle fixation with 0.5% PFA

  • Commercial kits optimized for intracellular/mitochondrial proteins

• Antibody Selection and Validation:

  • Choose antibodies validated for flow cytometry

  • Test fluorophore-conjugated antibodies or use secondary antibodies with bright fluorophores

  • Consider the size of fluorophore (smaller dyes may penetrate mitochondria better)

• Controls and Gating Strategy:

  • Use isotype controls at matching concentrations

  • Include TIMM9 knockdown cells as negative controls

  • Co-stain with mitochondrial markers (MitoTracker) to confirm localization

  • Gate on intact cells/mitochondria based on scatter properties

Specialized Applications:

Flow cytometric analysis can provide quantitative data on TIMM9 expression at the single-cell level, enabling researchers to investigate heterogeneity in expression and correlate with functional parameters.

How can CRISPR-Cas9 gene editing be combined with TIMM9 antibodies for functional studies?

Combining CRISPR-Cas9 gene editing with TIMM9 antibody applications creates powerful approaches for functional studies:

Experimental Design Strategies:

• TIMM9 Knockout/Knockdown Validation:

  • Generate TIMM9 knockout cell lines using CRISPR-Cas9

  • Validate knockout efficiency using TIMM9 antibodies in western blot, ICC, or flow cytometry

  • Create control cell lines with non-targeting gRNAs

• Structure-Function Analysis:

  • Introduce specific mutations (e.g., S49L variant) using CRISPR-Cas9 base editing

  • Use TIMM9 antibodies to assess protein expression, localization, and complex formation

  • Compare wildtype vs. mutant phenotypes in functional assays

• Tagged Endogenous TIMM9:

  • Insert epitope tags (FLAG, HA) or fluorescent proteins (GFP) at the TIMM9 locus

  • Use both tag-specific antibodies and TIMM9 antibodies to validate expression

  • Enable live cell imaging of TIMM9 dynamics

Functional Readouts:

• Mitochondrial respiration (Seahorse analyzer)

• Protein import efficiency (in vitro import assays)

• Cell proliferation and viability

• Metabolic profiling

• Tumor formation in xenograft models

This integrated approach allows for precise manipulation of TIMM9 and subsequent comprehensive analysis of functional consequences, providing insights into the mechanistic role of TIMM9 in mitochondrial function and cancer biology.

How can TIMM9 antibodies contribute to research on mitochondrial dysfunction in aging?

TIMM9 antibodies can provide valuable insights into the role of mitochondrial protein import in aging processes:

Research Applications:

• Age-Related Changes in Mitochondrial Import:

  • Compare TIMM9 expression and localization in tissues from young vs. aged organisms

  • Assess changes in TIMM9-containing complexes during aging

  • Investigate post-translational modifications of TIMM9 across the lifespan

• Intervention Studies:

  • Monitor TIMM9 expression in response to caloric restriction or exercise

  • Evaluate the impact of mitochondrial-targeted antioxidants on TIMM9 function

  • Assess TIMM9 changes in models of accelerated aging

• Single-Cell Analysis in Aging Tissues:

  • Apply TIMM9 antibodies in single-cell proteomics approaches

  • Investigate cell-type specific alterations in TIMM9 expression

  • Correlate with markers of cellular senescence

Methodological Approaches:

• Longitudinal studies using tissue samples from different age groups

• In vitro aging models (replicative senescence, stress-induced premature senescence)

• Comparative analysis across tissues with different metabolic demands

Understanding how TIMM9 and the mitochondrial import machinery change during aging could provide insights into interventions that might preserve mitochondrial function and delay age-related decline.

What novel applications of TIMM9 antibodies are emerging in precision medicine?

TIMM9 antibodies are finding novel applications in precision medicine approaches, particularly in cancer:

Emerging Applications:

• Companion Diagnostics:

  • Development of standardized IHC assays for TIMM9 expression

  • Potential stratification of patients for mitochondrial-targeted therapies

  • Correlation of TIMM9 expression with treatment response

• Liquid Biopsy Approaches:

  • Detection of TIMM9 protein in circulating tumor cells

  • Measurement of TIMM9 in extracellular vesicles

  • Correlation with disease progression and treatment response

• Combination Biomarker Panels:

  • Integration of TIMM9 with other mitochondrial markers

  • Development of risk assessment algorithms

  • Predictive models for metastasis and recurrence

Technical Innovations:

• Automated image analysis systems for standardized TIMM9 quantification

• Microfluidic devices for point-of-care TIMM9 detection

• AI-based interpretation of TIMM9 expression patterns