TIMMDC1 Antibody

What is TIMMDC1 and why is it important in mitochondrial research?

TIMMDC1, also known as C3orf1, is a predicted 4-pass membrane protein that localizes to the mitochondrial inner membrane. It functions as a chaperone protein involved in the assembly of the mitochondrial NADH:ubiquinone oxidoreductase complex (complex I) and specifically participates in constructing the membrane arm of this complex . TIMMDC1 is critical for maintaining proper mitochondrial respiration and ATP production, making it an important target for studying mitochondrial disorders, neurodegenerative diseases, and certain cancers .

What types of TIMMDC1 antibodies are available for research?

Available TIMMDC1 antibodies include polyclonal antibodies that target different epitopes of the protein. For example, some antibodies target amino acids 206-285 of human TIMMDC1 , while others may target different regions. These are available in various forms including unconjugated antibodies and those conjugated with FITC, HRP, or Biotin for different experimental applications . Commercial sources include Sigma (HPA053214), Santa Cruz (G-14), and others specialized for various applications such as Western blotting, immunohistochemistry, and ELISA .

What are the validated applications for TIMMDC1 antibodies?

TIMMDC1 antibodies have been validated for several applications including:

• Western blotting (WB) with recommended dilutions of 1:500 to 1:2000

• Immunohistochemistry (IHC) for tissue localization studies

• Enzyme-linked immunosorbent assay (ELISA) for quantitative analysis

• Immunoprecipitation (IP) in combination with mass spectrometry for protein interaction studies

What is the optimal protocol for Western blotting detection of TIMMDC1?

For optimal Western blotting results:

• Extract proteins from mitochondrial fractions or whole cells using appropriate lysis buffers

• Separate proteins using 4-16% gradient SDS-PAGE

• Transfer to PVDF membranes using standard protocols

• Block with 5% non-fat milk or BSA in TBST

• Incubate with primary TIMMDC1 antibody at dilutions between 1:500 and 1:2000

• Apply appropriate secondary antibody and develop using your detection system of choice

• The expected molecular weight for TIMMDC1 is approximately 33 kDa

For mitochondrial complex analysis, Blue Native PAGE (BN-PAGE) has been successfully used with 1% digitonin lysis of purified mitochondria .

How can I analyze TIMMDC1's role in mitochondrial complex I assembly?

To investigate TIMMDC1's role in complex I assembly:

• Purify mitochondria from control and TIMMDC1-depleted cells

• Lyse mitochondria in 1% digitonin

• Separate native complexes using 4-16% BN-PAGE

• Transfer to PVDF membranes

• Probe with antibodies against TIMMDC1 and other complex I components

• Quantify using densitometry to assess changes in complex formation

Alternative techniques include coupling SILAC (stable isotopic labeling by amino acids in culture) with size-based fractionation of purified mitochondria to obtain a quantitative view of complex I assembly and intermediates .

How should I design experiments to study TIMMDC1 knockdown effects on mitochondrial function?

When designing TIMMDC1 knockdown experiments:

• Use small interfering RNA (siRNA) or shRNA approaches targeting TIMMDC1

• Include appropriate controls (non-targeting sequences)

• Verify knockdown efficiency by Western blotting and qRT-PCR

• Assess mitochondrial complex I activity specifically, as TIMMDC1 knockdown has been shown to exclusively reduce the activity of complex I but not complexes II-IV

• Measure parameters of mitochondrial respiration and ATP-linked oxygen consumption using instruments like Seahorse XF Analyzer

• Examine glycolysis pathway activity as it may be secondarily affected

• Assess cellular phenotypes including proliferation, migration, and ATP content

This comprehensive approach will provide insights into both direct and indirect effects of TIMMDC1 depletion .

What controls should be included when validating TIMMDC1 antibody specificity?

To ensure antibody specificity:

• Include positive controls: tissues or cell lines known to express TIMMDC1 (most human cell lines express it at detectable levels)

• Include negative controls: TIMMDC1 knockdown or knockout samples

• Perform peptide competition assays to confirm epitope specificity

• Test cross-reactivity with similar proteins, particularly other TIM family members

• Validate results using multiple antibodies targeting different epitopes of TIMMDC1

• Include appropriate loading controls such as PCNA for nuclear fractions, HSP90 for cytosolic fractions, or TOMM70 for mitochondrial fractions

How can I optimize immunohistochemistry protocols for TIMMDC1 localization studies?

For optimal IHC results with TIMMDC1 antibodies:

• Test different fixation methods (formalin, paraformaldehyde, cold acetone)

• Optimize antigen retrieval techniques (heat-induced epitope retrieval in citrate buffer often works well for mitochondrial proteins)

• Use appropriate blocking solutions to reduce background

• Test a range of antibody dilutions

• Consider dual staining with other mitochondrial markers (TOMM20, MitoTracker, etc.) to confirm mitochondrial localization

• Include appropriate negative controls (antibody omission, isotype controls)

• Use counterstains that allow clear visualization of cellular structures without obscuring mitochondrial staining patterns

How can I investigate TIMMDC1 interactome and protein-protein interactions?

To study TIMMDC1 protein interactions:

• Perform immunoprecipitation using anti-TIMMDC1 antibodies followed by mass spectrometry analysis

• Consider crosslinking proteins prior to immunoprecipitation to capture transient interactions

• Use SILAC labeling for quantitative comparison of interactomes under different conditions

• Validate key interactions using reciprocal co-immunoprecipitation

• Perform BN-PAGE followed by second-dimension SDS-PAGE to separate complex I subassemblies

• Consider proximity labeling approaches such as BioID or APEX2 fused to TIMMDC1

Research has shown that TIMMDC1 reciprocally associates with multiple components of the ECSIT-TMEM126B-ACAD9-NDUFAF1 assembly factor complex (MCIA complex), as well as subunits of both the soluble and matrix arms of complex I .

How can I differentiate between the roles of TIMMDC1 in complex I assembly versus stability?

To distinguish assembly from stability functions:

• Perform time-course experiments following TIMMDC1 depletion

• Analyze complex I formation using pulse-chase labeling of mitochondrial proteins

• Use cycloheximide chase experiments to assess turnover rates of complex I subunits

• Analyze accumulation of subcomplexes through BN-PAGE and Western blotting

• Compare the effects of TIMMDC1 depletion on newly synthesized versus mature complex I

• Examine the effects of proteasome inhibitors on complex I subunits following TIMMDC1 depletion

Quantitative proteomics using SILAC has demonstrated that TIMMDC1 depletion leads to accumulation of characteristic CI subcomplexes, indicating its role in assembly .

What approaches can address contradictory results in TIMMDC1 studies?

When facing contradictory results:

• Consider cell type-specific differences (TIMMDC1 may have different importance in different tissues)

• Examine experimental conditions that might affect mitochondrial function (glucose vs. galactose media, oxygen levels)

• Compare acute (siRNA) versus chronic (shRNA, CRISPR) depletion methods

• Assess the efficiency of TIMMDC1 depletion as partial knockdown may yield different results

• Consider off-target effects of knockdown strategies

• Test different antibodies targeting distinct epitopes to ensure consistent detection

• Implement rescue experiments by expressing siRNA-resistant TIMMDC1 constructs to confirm specificity of observed phenotypes

How can TIMMDC1 antibodies be used to study mitochondrial disorders?

For investigating TIMMDC1 in mitochondrial disorders:

• Use TIMMDC1 antibodies to assess protein levels in patient-derived cells or tissues

• Perform immunohistochemistry to examine TIMMDC1 localization patterns in disease states

• Employ BN-PAGE to analyze complex I assembly defects in patient samples

• Correlate TIMMDC1 levels/localization with clinical severity and mitochondrial function parameters

• Evaluate the effects of therapeutic interventions on TIMMDC1 levels and complex I assembly

Research has identified a pathogenic intronic variant (c.597-1340A>G) in TIMMDC1 that enhances aberrant splicing and leads to almost complete loss of TIMMDC1 protein, resulting in compromised mitochondrial complex I function .

What is known about TIMMDC1's role in cancer progression and how can antibodies help study this?

TIMMDC1 has been implicated in cancer progression, particularly in gastric cancer:

• Use antibodies to compare TIMMDC1 expression between normal and cancerous tissues

• Assess correlation between TIMMDC1 levels and cancer stage/metastatic potential

• Perform immunohistochemistry on tissue microarrays to evaluate TIMMDC1 as a prognostic marker

• Investigate metabolic alterations in cancer cells following TIMMDC1 manipulation

Studies have shown that TIMMDC1 knockdown caused inhibitory effects on cell proliferation in vitro and tumor progression in vivo in gastric cancer models. The expression level of TIMMDC1 in highly-metastatic tumor cells is higher than in lowly-metastatic tumor cells, suggesting its potential as a therapeutic target .

How can TIMMDC1 antibodies be used to evaluate antisense oligonucleotide therapy effectiveness?

For therapeutic applications targeting TIMMDC1:

• Use antibodies to monitor TIMMDC1 protein restoration following splice-switching antisense oligonucleotide (SSO) treatment

• Perform Western blotting to quantify changes in TIMMDC1 levels before and after treatment

• Use immunocytochemistry to assess mitochondrial localization of restored TIMMDC1

• Couple with functional assays of complex I activity to confirm physiological relevance of protein restoration

Research has demonstrated successful restoration of normal TIMMDC1 mRNA processing and protein levels in patient cells using two different splice-switching antisense oligonucleotides, which also restored complex I subunit abundance and function as assessed by quantitative proteomics and real-time metabolic analysis .

What are the best methods for quantifying TIMMDC1 protein levels across experimental conditions?

For accurate quantification of TIMMDC1:

• Use Western blotting with appropriate loading controls (mitochondrial proteins like SDHA or TOMM70)

• Normalize TIMMDC1 signals to mitochondrial mass markers rather than whole-cell proteins

• Consider using fluorescence-based Western detection for wider linear range

• Implement ELISA techniques for more precise quantification

• Use mass spectrometry-based approaches for absolute quantification

• When comparing across cell types or tissues, consider differences in mitochondrial content

How should complex I activity data be correlated with TIMMDC1 expression levels?

To correlate TIMMDC1 levels with complex I function:

• Measure TIMMDC1 protein levels by Western blotting with densitometric analysis

• Assess complex I activity using standardized enzymatic assays (NADH:ubiquinone oxidoreductase activity)

• Measure oxygen consumption rates specifically linked to complex I using respiratory inhibitors

• Analyze BN-PAGE data to quantify fully assembled complex I versus subcomplexes

• Plot correlation graphs between TIMMDC1 levels and functional parameters

• Consider using regression analysis to determine the threshold of TIMMDC1 required for normal function

Research shows that TIMMDC1 knockdown significantly and exclusively reduces the activity of mitochondrial complex I but not complexes II-IV, causing inhibition in mitochondrial respiration and ATP-linked oxygen consumption .