TIMM50 Antibody

What is TIMM50 and why is it significant in mitochondrial research?

TIMM50 is a critical component of the mitochondrial import machinery, specifically functioning within the TIM23 complex alongside TIMM23 and TIMM17 (either TIMM17A or TIMM17B). This complex interacts with the TIMM44 component of the PAM complex and with DNAJC15, playing an essential role in the translocation of proteins across the inner mitochondrial membrane . TIMM50 contains an internal nuclear localization signal that can lead to nuclear localization in certain conditions, suggesting potential non-canonical functions beyond the mitochondria . Its significance extends to multiple pathological conditions, as dysregulation of TIMM50 has been implicated in cancer progression, including colorectal, breast, and non-small cell lung cancers .

What are the key considerations when selecting a TIMM50 antibody for research applications?

When selecting a TIMM50 antibody, researchers should consider: (1) Target epitope - different antibodies target various regions of TIMM50, such as amino acids 190-456, 396-409, or 74-104, which may affect recognition of specific isoforms or mutant variants ; (2) Host species - options include rabbit (most common), mouse, and goat, which impacts secondary antibody selection and potential cross-reactivity issues ; (3) Clonality - both monoclonal (e.g., clone 0W6J4, 6B1) and polyclonal options are available, offering different specificity and sensitivity profiles ; (4) Validated applications - ensure the antibody has been verified for your specific application (WB, IHC, IF, etc.) ; and (5) Species reactivity - confirm cross-reactivity with your experimental model, as some antibodies are human-specific while others also detect mouse or rat TIMM50 .

How do different TIMM50 epitopes influence antibody performance in various applications?

Different epitope-targeting antibodies demonstrate variable performance across applications due to epitope accessibility and conservation. Antibodies targeting amino acids 190-456 typically perform well in Western blot applications because this large region contains multiple antigenic determinants that remain accessible even after denaturation . Antibodies against the N-terminal region (AA 25-56 or 74-104) often excel in native-condition applications like immunoprecipitation where conformational epitopes remain intact . The C-terminal directed antibodies may demonstrate superior performance in immunohistochemistry applications depending on tissue fixation methods, as this region can remain accessible in formaldehyde-fixed tissues . When examining specific mutations or splice variants, researchers should select antibodies targeting regions distant from the variant site to ensure detection . For cross-species applications, antibodies targeting the most conserved regions (often in functional domains) offer the best performance across human, mouse, and rat samples .

What are the optimal conditions for using TIMM50 antibodies in Western blot applications?

For optimal Western blot results with TIMM50 antibodies, researchers should: (1) Load adequate protein (25 μg per lane is recommended based on validated protocols) ; (2) Use appropriate dilutions, typically 1:500-1:1000 for most TIMM50 antibodies ; (3) Block with 3% nonfat dry milk in TBST to minimize background ; (4) Use HRP-conjugated secondary antibodies at approximately 1:10000 dilution ; (5) Detect with standard ECL systems with exposure times around 30 seconds for clear visualization ; (6) Expect a band at approximately 50 kDa, though this may vary slightly depending on post-translational modifications and the specific cell type examined . When analyzing tissues with potentially variable TIMM50 expression, using GAPDH or another mitochondrial protein as a loading control is recommended for accurate quantification .

How should immunohistochemistry protocols be optimized for TIMM50 detection in tissue samples?

For effective TIMM50 immunohistochemistry, the protocol should include: (1) Tissue section preparation at 3 μm thickness for optimal antibody penetration ; (2) Heat-induced epitope retrieval using sodium citrate buffer (pH 6.0) at 95°C for 30 minutes to expose antigenic sites ; (3) Blocking of endogenous peroxidase activity with 3% hydrogen peroxide for 10 minutes at room temperature ; (4) Primary antibody incubation for 24 hours at manufacturer-recommended dilutions (typically 1:50-1:200) ; (5) Biotinylated secondary antibody incubation for 1 hour ; (6) Appropriate controls including positive controls (tissues with known TIMM50 expression) and negative controls (primary antibody omission) . When evaluating colorectal cancer tissues, a scoring system based on staining intensity and percentage of positive cells should be established to standardize interpretation, as TIMM50 overexpression has been correlated with pathologic stage, N stage, and distant metastasis .

What is the recommended approach for immunofluorescence applications using TIMM50 antibodies?

For immunofluorescence applications with TIMM50 antibodies, researchers should: (1) Use a dilution of 1:100 for most TIMM50 antibodies, which provides optimal signal-to-noise ratio ; (2) Include a mitochondrial co-staining marker (such as MitoTracker or TOM20 antibody) to confirm mitochondrial localization, as TIMM50 can occasionally demonstrate nuclear localization due to its internal nuclear localization signal ; (3) Use DAPI for nuclear counterstaining to precisely delineate nuclear boundaries and distinguish between mitochondrial and potential nuclear TIMM50 signals ; (4) Employ a high-magnification objective (40x or greater) to clearly resolve the characteristic mitochondrial network pattern ; (5) For studying TIMM50 mutations or alterations, compare staining patterns between normal and affected cells to identify any mislocalization or expression level differences . The mitochondrial network should show a typical reticular pattern in healthy cells, whereas cells with TIMM50 mutations may demonstrate altered mitochondrial morphology or TIMM50 distribution .

How can TIMM50 antibodies be used to investigate mitochondrial import defects in disease models?

TIMM50 antibodies can be leveraged to investigate mitochondrial import defects through: (1) Comparative immunoblotting of TIM23 complex components (TIMM23, TIMM17, TIMM44) in control versus disease models to assess stoichiometric changes in the import machinery ; (2) Co-immunoprecipitation using TIMM50 antibodies to evaluate interaction partners and identify altered protein associations in pathological conditions ; (3) Immunofluorescence co-localization studies with mitochondrial matrix proteins to quantify import efficiency ; (4) Pulse-chase experiments combining TIMM50 immunoprecipitation with radiolabeled precursor proteins to directly measure import kinetics ; (5) Proximity labeling approaches using TIMM50 antibodies to identify the changing interaction landscape in disease states . Research on TIMM50 mutations has revealed significant decreases in TIM23 core protein levels, affecting the import of multiple proteins involved in calcium homeostasis (MICU2, SLC25A3, LETM1), heme synthesis (PPOX, CPOX), and cardiolipin synthesis (HADHA), while proteins involved in Fe-S cluster biosynthesis, detoxification, and amino acid metabolism remained largely unaffected .

What methodologies exist for studying TIMM50's role in cancer progression using available antibodies?

Advanced methodologies for studying TIMM50's role in cancer include: (1) Tissue microarray analysis using validated TIMM50 antibodies to correlate expression levels with clinicopathological features and patient outcomes across large cohorts ; (2) Chromatin immunoprecipitation followed by sequencing (ChIP-seq) to identify potential transcriptional regulatory roles when TIMM50 localizes to the nucleus ; (3) Proximity-dependent biotin identification (BioID) combined with TIMM50 antibody validation to map the changing protein interaction network in cancer versus normal cells ; (4) Quantitative phosphoproteomics following TIMM50 knockdown or overexpression to elucidate its role in signaling cascades, such as the ERK/P90RSK pathway implicated in non-small cell lung cancer ; (5) Immunohistochemical multiplex staining to simultaneously visualize TIMM50 alongside markers of proliferation, apoptosis, and cancer stemness . Research has demonstrated that TIMM50 overexpression promotes tumor progression in multiple cancer types, with studies in colorectal cancer showing significant associations between high TIMM50 expression and advanced pathologic stage, lymph node metastasis, and distant metastasis .

How can TIMM50 antibodies be employed to study mitochondrial dysfunction in neurological disorders?

For investigating neurological disorders, TIMM50 antibodies can be utilized in these sophisticated approaches: (1) Immunohistochemical analysis of post-mortem brain tissue comparing TIMM50 expression and localization patterns between control and diseased samples ; (2) Triple immunofluorescence labeling of primary neuronal cultures combining TIMM50 antibodies with markers for mitochondrial membrane potential and reactive oxygen species to correlate protein expression with functional parameters ; (3) Super-resolution microscopy with validated TIMM50 antibodies to examine mitochondrial ultrastructure and protein complex organization at nanoscale resolution ; (4) Electron microscopy immunogold labeling to precisely localize TIMM50 within mitochondrial compartments in neuronal tissues ; (5) Live-cell imaging in neuronal models utilizing fluorescently-tagged antibody fragments to track dynamic changes in TIMM50 localization during cellular stress . Recent research has demonstrated that TIMM50 mutations can significantly impact neuronal excitability through mechanisms that appear related to altered mitochondrial protein import, particularly affecting calcium homeostasis proteins and potentially influencing ion channel expression or function in the plasma membrane .

What are the common challenges encountered when using TIMM50 antibodies and how can they be addressed?

Common challenges with TIMM50 antibodies include: (1) Nonspecific binding in Western blots, which can be mitigated by increasing blocking time with 5% BSA or milk, optimizing antibody dilution, and including appropriate detergents in wash buffers ; (2) Weak signals in immunostaining applications, which may be improved through enhanced antigen retrieval methods such as pressure cooking in citrate buffer (pH 6.0) or using EDTA buffer (pH 9.0) ; (3) Background staining in tissue samples, which can be reduced by pre-absorbing the primary antibody with non-specific proteins or using more stringent washing protocols ; (4) Inconsistent results between different antibody clones, which necessitates validation with multiple antibodies targeting different epitopes and correlation with mRNA expression data ; (5) Cross-reactivity with related proteins, which can be controlled by performing siRNA knockdown controls to confirm specificity . For TIMM50 specifically, researchers should be aware that its dual localization (mitochondrial and occasionally nuclear) may complicate interpretation of staining patterns, requiring careful co-localization studies with compartment-specific markers .

What validation steps should be performed before using a new TIMM50 antibody in critical experiments?

Before using a new TIMM50 antibody in critical experiments, researchers should perform these validation steps: (1) Western blot analysis across multiple cell lines or tissues to confirm the antibody detects a protein of the expected molecular weight (~50 kDa) ; (2) Comparison with mRNA expression levels using RT-qPCR with validated primers (such as forward: 5'-TTCCTGATGAGTTCGACAATG-3' and reverse: 5'-AGCTCCAAAACGAGCGTGTA-3') ; (3) RNA interference or CRISPR-based knockout controls to confirm signal specificity, with expected reduction or elimination of signal ; (4) Peptide competition assay using the immunizing peptide to demonstrate binding specificity ; (5) Comparison of results across multiple TIMM50 antibodies targeting different epitopes to confirm consistent patterns . Additionally, researchers studying disease-associated mutations should verify that the antibody's epitope does not overlap with mutation sites that might affect recognition . For functional studies, validation should include evidence that the antibody does not interfere with TIMM50's interactions with other TIM23 complex components when used in non-denaturing applications .

How can researchers quantitatively assess TIMM50 expression levels in different experimental systems?

For quantitative assessment of TIMM50 expression, researchers should employ: (1) Quantitative Western blotting with infrared or chemiluminescent detection systems using standard curves of recombinant TIMM50 for absolute quantification ; (2) RT-qPCR with validated primers and GAPDH normalization (as used in published studies) for mRNA quantification ; (3) Quantitative immunofluorescence with automated image analysis software measuring integrated density of TIMM50 signal normalized to mitochondrial mass ; (4) Flow cytometry with permeabilized cells and validated TIMM50 antibodies for high-throughput single-cell analysis of expression levels ; (5) ELISA-based approaches for sample types where maintaining protein conformation is important . When comparing expression across different pathological states, such as cancer progression, researchers should establish a standardized scoring system based on both staining intensity and percentage of positive cells, as has been done in colorectal cancer research where TIMM50 expression correlated with disease stage and metastatic status . For mitochondrial dysfunction studies, TIMM50 levels should be evaluated in relation to other mitochondrial proteins to distinguish between specific TIMM50 alterations and general changes in mitochondrial content .

How should researchers interpret changes in TIMM50 protein levels in disease models compared to controls?

When interpreting altered TIMM50 expression in disease models, researchers should consider: (1) Whether changes are specific to TIMM50 or reflect broader alterations in mitochondrial content by comparing with other mitochondrial markers such as TOM20 or VDAC ; (2) The potential impact on TIM23 complex stoichiometry by analyzing ratios of TIMM50 to other complex components like TIMM23 and TIMM17 ; (3) Correlation between TIMM50 protein changes and corresponding mRNA levels to distinguish between transcriptional and post-transcriptional regulation ; (4) Subcellular distribution shifts that might indicate altered function, particularly between mitochondrial and nuclear localization ; (5) Relationship to downstream functional consequences such as changes in mitochondrial membrane potential, protein import efficiency, or respiratory chain activity . In cancer research, TIMM50 overexpression has been linked to tumor progression in multiple cancer types, suggesting potential oncogenic properties . Conversely, in neurological disorders associated with TIMM50 mutations, decreased functional protein levels may lead to mitochondrial dysfunction affecting specific protein import pathways, with selective effects on proteins involved in calcium homeostasis and certain biosynthetic pathways, while leaving other import pathways relatively intact .

What insights have TIMM50 antibody-based studies provided into mitochondrial disease mechanisms?

TIMM50 antibody-based studies have revealed crucial insights into mitochondrial disease mechanisms: (1) Characterization of TIMM50 mutations in patient fibroblasts has demonstrated that TIMM50 deficiency leads to selective rather than global defects in mitochondrial protein import ; (2) Proteomic analyses using TIMM50 antibodies have identified specific protein cohorts affected by TIMM50 dysfunction, particularly those involved in calcium homeostasis (MICU2, SLC25A3, LETM1), heme synthesis (PPOX, CPOX), and cardiolipin synthesis (HADHA) ; (3) Surprisingly, matrix proteins involved in Fe-S cluster biosynthesis, detoxification, fatty acid oxidation, and many TCA cycle components remained unaffected by TIMM50 mutations ; (4) Unexpectedly, some matrix proteins (ALDH2, GRSF-1, AK4, LACTB2, OAT) showed increased steady-state levels in patient fibroblasts, with ALDH2 and GRSF-1 demonstrating 15-fold and 6-fold increases respectively ; (5) These findings suggest that TIMM50 dysfunction may trigger compensatory mechanisms for specific mitochondrial pathways rather than causing uniform mitochondrial failure . These insights contribute to our understanding of the selective vulnerability of certain tissues and cellular functions in mitochondrial disorders, potentially explaining why some mitochondrial diseases present with tissue-specific manifestations despite the ubiquitous expression of affected proteins .

How do TIMM50 expression patterns correlate with clinical outcomes in cancer research?

TIMM50 expression correlations with cancer outcomes reveal significant clinical relevance: (1) In colorectal cancer, immunohistochemical studies using TIMM50 antibodies have demonstrated that high TIMM50 expression significantly associates with advanced pathologic stage, lymph node metastasis (N stage), and distant metastasis ; (2) TIMM50 overexpression has been identified as an independent prognostic factor in multiple cancer types, with elevated expression correlating with poorer survival outcomes ; (3) Studies in non-small cell lung cancer have revealed that TIMM50 promotes tumor progression through phosphorylation of the ERK/P90RSK pathway, suggesting its potential as both a prognostic marker and therapeutic target ; (4) In breast cancer research, TIMM50 antibody-based investigations have shown that loss of TIMM50 suppresses cancer cell proliferation and induces apoptosis, indicating its pro-survival role in malignant cells ; (5) The nuclear localization of TIMM50, detectable with specific antibodies and co-localization studies, may represent a particular cancer-associated phenomenon with distinct prognostic implications compared to exclusively mitochondrial localization . These findings collectively support the potential utility of TIMM50 as a sensitive biomarker for patient stratification and prognostication in oncology, with possible implications for personalized treatment approaches targeting mitochondrial function in cancer cells .