MTIF3 Antibody

Basic Research Questions

• What is the recommended dilution range for MTIF3 antibodies in Western blot applications?

  MTIF3 antibodies typically perform optimally at dilutions between 1:500-1:2000 for Western blot applications. For example, the Proteintech antibody (14219-1-AP) has been validated at this range, while the Boster antibody (A11221-1) shows similar dilution requirements . When first working with an MTIF3 antibody, it's advisable to perform a dilution series to determine the optimal concentration for your specific experimental conditions and cell/tissue type, as background signal can vary between samples.

  Application	Recommended Dilution
  Western Blot	1:500-1:2000
  Immunoprecipitation	0.5-4.0 μg for 1.0-3.0 mg of total protein lysate
  Immunohistochemistry	1:50-1:500

• What is the expected molecular weight for MTIF3 in Western blot analysis?

  While the calculated molecular weight of MTIF3 is approximately 32 kDa, the observed molecular weight in Western blot analysis is typically around 29 kDa . This discrepancy between calculated and observed molecular weights is common for many proteins and may be attributed to post-translational modifications, protein folding, or the specific migration properties of the protein in SDS-PAGE. When validating MTIF3 antibodies, researchers should look for a primary band at approximately 29 kDa, though the exact position may vary slightly depending on the experimental conditions and gel percentage used.

• Which cell lines and tissues show reliable MTIF3 expression for antibody validation?

  Several cell lines and tissues have demonstrated consistent MTIF3 expression suitable for antibody validation:

  Cell Lines	Tissues
  HeLa (human cervical cancer)	Human liver
  HepG2 (human liver cancer)	Human prostate
  A431 (human epidermoid carcinoma)	Mouse testis
  HT-29 (human colorectal adenocarcinoma)	Human testis
  Human preadipocyte cells	Mouse heart

  These samples have been experimentally verified to express detectable levels of MTIF3 and can serve as positive controls for antibody validation . For initial antibody characterization, HeLa cells are particularly recommended as they consistently show good expression levels.

• What antigen retrieval methods are recommended for MTIF3 immunohistochemistry?

  For optimal MTIF3 detection in immunohistochemistry applications, the suggested antigen retrieval method involves TE buffer at pH 9.0. Alternatively, citrate buffer at pH 6.0 can also be used, though possibly with reduced sensitivity . This is particularly important when working with formalin-fixed, paraffin-embedded (FFPE) tissues, as proper antigen retrieval significantly impacts staining quality. The specific protocol has been validated with human liver tissue, mouse testis tissue, and human testis tissue, all showing reliable MTIF3 immunoreactivity following appropriate antigen retrieval.

Advanced Research Questions

• How can MTIF3 antibodies be used to investigate mitochondrial translation in neuronal axons?

  To study mitochondrial translation in axons using MTIF3 antibodies, researchers should consider microfluidic chamber approaches that physically separate axons from cell bodies. As demonstrated in recent studies, BDNF (brain-derived neurotrophic factor) induces local translation of MTIF3 mRNA in axonal growth cones .

  Methodological approach:

  • Culture primary neurons in microfluidic chambers to isolate axonal compartments

  • Apply treatments (e.g., BDNF, translation inhibitors) selectively to axonal chambers

  • Use MTIF3 antibodies in combination with proximity ligation assays or bimolecular fluorescence complementation (BiFC) techniques to visualize newly synthesized MTIF3

  • For quantification, compare MTIF3 signals in axons under different conditions (e.g., with/without BDNF treatment)

  • Include cycloheximide controls to distinguish between locally synthesized MTIF3 and protein transported from the cell body

  Research has shown that BDNF-induced mito-riboBiFC signals in axon growth cones are blocked by cycloheximide treatment, confirming that local protein synthesis promotes mitochondrial translation through MTIF3 .

• What strategies can be employed to study the role of MTIF3 in OXPHOS complex assembly?

  To investigate MTIF3's role in OXPHOS complex assembly, researchers should combine MTIF3 manipulation (knockout/knockdown) with Blue Native-PAGE analysis of mitochondrial complexes.

  Experimental approach:

  • Generate MTIF3-deficient cells using CRISPR-Cas9 or RNA interference

  • Isolate intact mitochondria using differential centrifugation

  • Analyze OXPHOS complex assembly using Blue Native-PAGE

  • Perform western blot analysis with antibodies against specific complex subunits

  • Quantify assembly differences between control and MTIF3-deficient cells

  Recent studies have demonstrated that MTIF3 deficiency leads to decreased complex III₂/IV₂ and IV₁ assembly, with trending decreased complex V/III₂+IV₁ assembly . Interestingly, OXPHOS complex II assembly was significantly increased in MTIF3 knockout cells, suggesting compensatory mechanisms. These findings indicate that MTIF3 plays a crucial role in the proper assembly of respiratory chain complexes.

• How can researchers assess the impact of MTIF3 variants on mitochondrial function?

  To evaluate how MTIF3 genetic variants affect mitochondrial function, researchers should implement a comprehensive approach combining genetic editing, expression analysis, and functional assays.

  Methodological workflow:

  • Use CRISPR-Cas9 to edit potential functional variants (e.g., rs67785913)

  • Confirm altered MTIF3 expression using qRT-PCR and western blotting

  • Assess mitochondrial function through:

    • Seahorse Mito Stress Tests to measure oxygen consumption rate (OCR)

    • Analysis of mitochondrial DNA-encoded protein expression

    • Mitochondrial DNA content quantification

    • Fatty acid oxidation assays

    • Evaluation of triglyceride retention under glucose restriction

  Studies have shown that cells with CRISPR-edited rs67785913 CTCT allele exhibit significantly higher MTIF3 expression than cells with the rs67785913 CT allele . Functional analyses revealed that MTIF3 deficiency leads to reduced mitochondrial respiration, altered expression of mitochondrial DNA-encoded genes and proteins, and disturbed OXPHOS complex assembly. Furthermore, under glucose restriction, MTIF3 knockout cells retained more triglycerides than control cells, highlighting MTIF3's role in lipid metabolism.

• What methodological approaches are optimal for studying MTIF3's role in neurodegeneration?

  To investigate MTIF3's involvement in neurodegenerative conditions like Parkinson's disease, researchers should employ a multi-faceted approach:

  Experimental strategy:

  • Genotype patients and controls for MTIF3 polymorphisms (particularly rs7669)

  • Assess MTIF3 mRNA expression levels using qRT-PCR in relevant cell types

  • Perform case-control association studies with adequate sample sizes

  • Use patient-derived cells to analyze mitochondrial function

  • Employ MTIF3 antibodies to assess protein expression and localization

  Research has demonstrated a significant association between the combined TT/CT-genotypes of rs7669 versus the CC-genotype with Parkinson's disease (P = 0.0473) . Furthermore, the TT-genotype causes a significant decrease in MTIF3 mRNA expression compared to the CC-genotype (P = 0.0163), suggesting a functional impact of this synonymous polymorphism on MTIF3 expression and potentially contributing to disease pathogenesis.

• How can MTIF3 antibodies be utilized to study the protein's role in adipocyte metabolism?

  To investigate MTIF3's function in adipocyte metabolism, researchers should implement the following methodological approach:

  Experimental design:

  • Generate MTIF3 knockouts in differentiated human adipocytes

  • Validate knockout efficiency using MTIF3 antibodies in western blot

  • Assess adipocyte differentiation markers (PPARG, ADIPOQ, CEBPA, SREBF1, FABP4)

  • Analyze mitochondrial function through:

    • Seahorse assays for mitochondrial respiration

    • Oil-Red O staining for lipid accumulation

    • Triglyceride content quantification

    • Metabolomic profiling under normal and glucose-restricted conditions

  Recent studies have established that MTIF3 plays a crucial role in adipocyte mitochondrial function and lipid metabolism . MTIF3-deficient adipocytes show reduced mitochondrial respiration and endogenous fatty acid oxidation, with altered expression of mitochondrial DNA-encoded genes and proteins. Additionally, after glucose restriction, MTIF3 knockout cells retained more triglycerides than control cells, demonstrating an adipocyte-specific role of MTIF3 in lipid metabolism and potentially explaining its association with obesity and response to weight loss interventions.

  Analysis	Findings in MTIF3 Knockout Adipocytes
  Mitochondrial proteins	Decreased COX II, ND2; trending decrease of CYTB
  Mitochondrial gene expression	Higher expression of MT-ND1, MT-ND2; lower expression of MT-ND3, MT-CO3
  Mitochondrial DNA content	Significantly reduced
  OXPHOS complex assembly	Decreased complex III₂/IV₂ and IV₁ assembly; increased complex II assembly
  Lipid metabolism	Increased triglyceride retention after glucose restriction

• What techniques can distinguish between local translation and transport of MTIF3 in neurons?

  To differentiate between locally translated MTIF3 and protein transported from the neuronal cell body, researchers should employ compartmentalized culture systems combined with translation inhibitors and protein labeling techniques:

  Methodological approach:

  • Use microfluidic devices to physically separate axons from cell bodies

  • Apply translation inhibitors (cycloheximide or anisomycin) selectively to axonal compartments

  • Employ puromycin labeling (SUnSET technique) to visualize newly synthesized proteins

  • Use proximity ligation assays to detect newly synthesized MTIF3

  • Implement bimolecular fluorescence complementation (BiFC) sensors to monitor mitochondrial translation

  Studies have shown that BDNF-induced mito-riboBiFC signals in axon growth cones are completely blocked by cycloheximide treatment in the axonal compartment, confirming that local protein synthesis promotes mitochondrial translation through MTIF3 . These findings demonstrate that MTIF3 is locally translated in response to BDNF and subsequently facilitates mitochondrial translation in distal axons, supporting axonal development.

Research Design Considerations

• What controls are essential when performing MTIF3 knockout or knockdown experiments?

  When conducting MTIF3 manipulation studies, the following controls are critical:

  • Off-target assessment: Perform T7EI assays on PCR-amplified top predicted off-target sites (as demonstrated in MTIF3 knockout studies where no detectable off-targeting was observed)

  • Expression validation: Confirm MTIF3 reduction at both mRNA (qRT-PCR) and protein (western blot) levels

  • Phenotypic controls: Assess whether knockout affects basic cellular processes that might confound specific mitochondrial phenotypes

  • Rescue experiments: Reintroduce wild-type MTIF3 to confirm phenotype specificity

  • Appropriate control constructs: Use scrambled control or empty vector controls matched to the experimental system

  In published MTIF3 knockout studies, researchers achieved >80% reduction in MTIF3 protein levels and confirmed that the knockout did not affect adipogenic markers (ACC, FABP4, FAS), ensuring that observed phenotypes were specific to mitochondrial function rather than adipogenic differentiation .

• How should researchers design experiments to study MTIF3's role in gene-environment interactions related to obesity?

  To investigate MTIF3's involvement in gene-environment interactions affecting obesity, researchers should implement a comprehensive experimental design:

  Methodological framework:

  • Genotype participants for relevant MTIF3 variants (particularly rs67785913)

  • Stratify analyses by genotype to identify potential gene-diet interactions

  • In cellular models:

    • Create isogenic cell lines differing only in the relevant MTIF3 variant

    • Subject cells to conditions mimicking dietary interventions (glucose restriction, lipid challenges)

    • Assess mitochondrial function, lipid metabolism, and gene expression

  • For human studies:

    • Design intervention studies stratified by MTIF3 genotype

    • Measure mitochondrial parameters in accessible tissues (e.g., blood cells, adipose biopsies)

    • Correlate genotype with intervention response

  Research has demonstrated that after glucose restriction, MTIF3 knockout cells retain more triglycerides than control cells, suggesting that MTIF3 may influence adipocyte response to caloric restriction . Additionally, genetic variants that increase MTIF3 expression (e.g., the rs67785913 CTCT allele) may be associated with better response to weight loss interventions, representing a true gene-environment interaction.