AIFM2 Antibody, HRP conjugated

What is AIFM2 and what cellular functions does it regulate?

AIFM2 (Apoptosis Inducing Factor Mitochondria Associated 2) is a flavoprotein oxidoreductase that functions as a crucial NADH oxidase involved in regulating cytosolic NAD+ levels . This protein binds single-stranded DNA and contributes to apoptosis when bacterial or viral DNA is present . Additionally, AIFM2 plays significant roles in:

• Apoptotic pathways as part of the FAD-dependent oxidoreductase protein family

• NAD+/NADH ratio regulation, which directly impacts glycolytic metabolism

• Mitochondrial biogenesis and oxidative phosphorylation through the SIRT1/PGC-1α signaling pathway

• Cancer progression, particularly in hepatocellular carcinoma where it promotes metastasis

AIFM2 is also known by several synonyms including AMID, Apoptosis inducing factor (AIF) homologous mitochondrion associated inducer of death, and Ferroptosis suppressor protein 1 (FSP1) .

Where is AIFM2 predominantly localized within cells?

AIFM2 displays multiple subcellular localizations which are important considerations when designing immunodetection experiments. According to available research data, AIFM2 can be found in:

• Cytoplasm

• Mitochondrion outer membrane

• Cell membrane

• Nucleus

This diverse localization pattern requires careful experimental design when using AIFM2 antibodies to ensure proper visualization of the protein in its native compartments. When using immunohistochemistry techniques, researchers should consider appropriate cell permeabilization methods to access these various cellular compartments.

What tissue types show notable AIFM2 expression patterns?

• Hepatocellular carcinoma (HCC)

• Lung adenocarcinoma (LUAD)

• Stomach and Esophageal carcinoma (STES)

• Kidney renal papillary cell carcinoma (KIRP)

• Stomach adenocarcinoma (STAD)

• Uterine Corpus Endometrial Carcinoma (UCEC)

• Kidney Chromophobe (KICH)

• Cholangiocarcinoma (CHOL)

When designing experiments with AIFM2 antibodies, researchers should consider this expression profile to select appropriate positive and negative control tissues.

How can AIFM2 antibodies be utilized to investigate metabolic regulation in skeletal muscle?

AIFM2 antibodies can be instrumental in studying the protein's role in muscle metabolism, particularly given recent findings about its impact on glycolysis and NAD+/NADH ratios. A methodological approach involves:

• Implement immunoblotting with anti-AIFM2 antibodies to quantify expression levels in glycolytic versus oxidative muscle fibers

• Use immunofluorescence microscopy with anti-AIFM2 antibodies to visualize protein localization relative to mitochondria and other metabolic organelles

• Couple AIFM2 detection with measurement of NAD+/NADH ratios using biochemical assays (similar to the lactate dehydrogenase cycling approach)

• Correlate AIFM2 expression with extracellular acidification rate (ECAR) measurements to assess glycolytic flux

• Compare these parameters between sedentary and exercise-trained muscle samples to elucidate AIFM2's role in exercise adaptation

Research indicates that AIFM2 overexpression in C2C12 myotubes results in approximately 2.8-fold increase in NAD+/NADH ratio compared to control cells, with corresponding increases in both basal and maximal extracellular acidification rates, suggesting enhanced glycolytic capacity .

What methodologies are most effective for studying AIFM2's role in cancer progression using antibody-based techniques?

Based on recent research into AIFM2's role in hepatocellular carcinoma, a multi-faceted approach using AIFM2 antibodies can provide valuable insights:

• Perform immunohistochemistry (IHC) on paired tumor and adjacent non-tumor tissues to quantify expression differences

• Use Western blot analysis with anti-AIFM2 antibodies to validate expression changes in tumor versus normal cell lines

• Implement co-immunoprecipitation with AIFM2 antibodies to identify protein-protein interactions, particularly with PGC-1α and SIRT1

• Combine AIFM2 antibody staining with markers of mitochondrial biogenesis to assess correlation between AIFM2 expression and mitochondrial function

• Utilize tissue microarrays with AIFM2 antibodies for high-throughput analysis of expression across multiple cancer types

Research has demonstrated that higher AIFM2 expression correlates with poorer patient survival and increased risk of recurrence in HCC, suggesting its value as a prognostic marker .

How can researchers effectively study the relationship between AIFM2 and mitochondrial biogenesis?

To investigate AIFM2's influence on mitochondrial biogenesis, researchers can implement the following methodological approach:

• Use AIFM2 antibodies for immunofluorescence co-localization studies with mitochondrial markers

• Perform subcellular fractionation followed by Western blotting with AIFM2 antibodies to quantify protein distribution between cytosolic and mitochondrial fractions

• Measure changes in oxygen consumption rate (OCR) and oxidative phosphorylation (OXPHOS) complex activities following AIFM2 manipulation

• Implement chromatin immunoprecipitation (ChIP) assays to study AIFM2's impact on transcription factors regulating mitochondrial genes

• Analyze correlation between AIFM2 and PGC-1α protein levels using dual immunohistochemistry staining

Research indicates that AIFM2 promotes mitochondrial biogenesis through post-transcriptional upregulation of PGC-1α, with a significant positive correlation between their expression levels in HCC tissues .

What are the optimal dilution ratios and applications for AIFM2 antibodies in different experimental contexts?

Based on available research data, the following application-specific parameters are recommended:

When working with HRP-conjugated AIFM2 antibodies specifically, researchers should:

• Perform a dilution series to determine optimal antibody concentration

• Include appropriate positive controls (tissues with known AIFM2 expression)

• Consider longer incubation times at lower antibody concentrations to improve signal-to-noise ratio

• Optimize blocking conditions to minimize background, particularly when using HRP conjugates

What are common technical challenges when working with AIFM2 antibodies and how can they be addressed?

When using AIFM2 antibodies, researchers may encounter several technical issues that require troubleshooting:

• Non-specific binding:

  • Problem: Background staining obscuring specific AIFM2 signal

  • Solution: Use more stringent blocking (5% BSA or 5% non-fat milk) and include 0.1-0.3% Triton X-100 in antibody diluent for better penetration

• Inconsistent signal across experiments:

  • Problem: Variable staining intensity between replicates

  • Solution: Standardize fixation protocols, maintain consistent antibody lot numbers, and prepare fresh working solutions for each experiment

• Weak signal in mitochondrial fractions:

  • Problem: Difficulty detecting AIFM2 in mitochondrial outer membrane

  • Solution: Optimize mitochondrial isolation protocols and consider membrane solubilization methods appropriate for outer membrane proteins

• Multiple bands on Western blot:

  • Problem: Detection of multiple bands that may represent isoforms or degradation products

  • Solution: Validate with recombinant AIFM2 positive control and consider using gradient gels for better separation

• Cross-reactivity concerns:

  • Problem: Antibody binding to other FAD-dependent oxidoreductase family members

  • Solution: Use AIFM2 knockout/knockdown samples as negative controls to confirm specificity

How should researchers design controls for AIFM2 antibody experiments?

Proper control design is essential for generating reliable data with AIFM2 antibodies:

• Positive Controls:

  • Include tissues/cells with known high AIFM2 expression (colorectal cancer, hepatocellular carcinoma)

  • Consider using recombinant AIFM2 protein as a standard for Western blot

  • Include AIFM2 overexpression samples when available

• Negative Controls:

  • Use AIFM2 knockdown or knockout cell lines to validate specificity

  • Include isotype control antibodies at equivalent concentrations

  • For HRP-conjugated antibodies, perform experiments with HRP-conjugated isotype controls

• Method-Specific Controls:

  • For IHC: Include no-primary-antibody controls and non-specific IgG controls

  • For WB: Include molecular weight markers and loading controls

  • For IF: Include secondary-antibody-only controls and autofluorescence controls

• Validation Across Methods:

  • Confirm findings using multiple detection methods (e.g., validate IHC findings with Western blot)

  • Use multiple antibodies targeting different epitopes when possible

  • Compare results between polyclonal and monoclonal antibodies when available

How can researchers quantitatively analyze AIFM2 expression patterns in relation to disease progression?

Quantitative analysis of AIFM2 expression requires systematic approaches:

• Immunohistochemistry Scoring:

  • Implement standardized scoring systems (e.g., H-score, Allred score) for semiquantitative analysis

  • Use digital pathology software to quantify staining intensity and percentage of positive cells

  • Consider multiplexed IHC to correlate AIFM2 with other markers in the same tissue section

• Survival Analysis Methodology:

  • Stratify patients into high and low AIFM2 expression groups based on median expression or optimal cutoff values

  • Perform Kaplan-Meier survival analysis to correlate expression with patient outcomes

  • Use Cox proportional hazards models to adjust for confounding variables

• Correlation with Clinicopathological Features:

  • Create correlation matrices between AIFM2 expression and features like tumor size, stage, and metastasis

  • Use appropriate statistical tests (Chi-square, Fisher's exact test, etc.) based on data distribution

  • Present findings in comprehensive tables showing statistical significance

How should researchers interpret AIFM2 expression changes in the context of the NAD+/NADH ratio and cellular metabolism?

To accurately interpret AIFM2's impact on cellular metabolism:

• Integrated Analysis Approach:

  • Measure AIFM2 protein levels in parallel with NAD+/NADH ratio using biochemical assays

  • Correlate with measurements of glycolytic flux (ECAR) and oxidative phosphorylation (OCR)

  • Analyze expression of related metabolic enzymes to establish pathway relationships

• Experimental Validation:

  • Compare wild-type cells to those with AIFM2 overexpression or knockdown

  • Measure changes in NAD+/NADH ratio, SIRT1 activity, and PGC-1α expression levels

  • Use metabolic inhibitors to block specific pathways and determine AIFM2's point of action

• Tissue-Specific Considerations:

  • Compare AIFM2's metabolic effects between glycolytic and oxidative tissues

  • Account for baseline metabolic differences between normal and cancer tissues

  • Analyze changes in relation to tissue-specific expression of metabolic regulators

Research has shown that AIFM2 overexpression results in significantly increased NAD+/NADH ratios (approximately 2.8-fold) and enhanced glycolytic capacity in muscle cells , while in cancer cells it promotes oxidative phosphorylation through activation of SIRT1/PGC-1α signaling .

What are the important considerations when interpreting data about AIFM2's role in different subcellular compartments?

When analyzing AIFM2's distribution and function across cellular compartments:

• Subcellular Fractionation Quality Control:

  • Verify fractionation purity using established markers for different compartments

  • Account for potential cross-contamination between fractions

  • Consider dynamic redistribution of AIFM2 under different cellular conditions

• Co-localization Analysis:

  • Use high-resolution microscopy (confocal, super-resolution) to accurately determine spatial relationships

  • Implement quantitative co-localization metrics (Pearson's coefficient, Manders' overlap)

  • Consider three-dimensional analysis to account for the full cellular volume

• Functional Context:

  • Correlate localization patterns with known functions in each compartment

  • Consider how mitochondrial outer membrane localization relates to apoptotic function

  • Analyze nuclear localization in relation to possible transcriptional regulation roles

• Dynamic Trafficking Analysis:

  • Investigate potential translocation of AIFM2 between compartments under stress conditions

  • Consider post-translational modifications that might regulate localization

  • Analyze temporal changes in localization patterns following experimental manipulations

AIFM2 has been reported in multiple cellular locations including the cytoplasm, mitochondrial outer membrane, cell membrane, and nucleus , suggesting compartment-specific functions that require careful experimental design to elucidate.

What emerging research areas could benefit from AIFM2 antibody applications?

Based on current understanding of AIFM2 functions, several promising research directions emerge:

• Cancer Metabolism and Therapy Resistance:

  • Investigate AIFM2's role in metabolic reprogramming of cancer cells

  • Explore potential connections between AIFM2 expression and resistance to specific therapies

  • Develop combination approaches targeting AIFM2-dependent metabolic pathways

• Exercise Physiology and Muscle Adaptation:

  • Examine AIFM2's involvement in skeletal muscle adaptation to exercise training

  • Investigate fiber-type specific expression patterns and functional consequences

  • Explore potential applications in improving exercise performance or recovery

• Neurodegenerative Diseases:

  • Study AIFM2's role in neuronal metabolism and mitochondrial function

  • Investigate potential connections to neurodegenerative processes

  • Explore therapeutic targeting of AIFM2 pathways in neurological conditions

• Aging and Senescence:

  • Analyze age-related changes in AIFM2 expression and function

  • Investigate connections to the NAD+/NADH ratio decline observed in aging

  • Explore potential interventions targeting AIFM2 to combat age-related metabolic decline

Each of these research areas would benefit from advanced applications of AIFM2 antibodies, particularly HRP-conjugated variants that offer enhanced sensitivity for detection in various experimental contexts.

How might researchers integrate AIFM2 antibody techniques with advanced molecular and imaging methods?

Future research could leverage AIFM2 antibodies in combination with cutting-edge techniques:

• Spatial Transcriptomics and Proteomics:

  • Combine AIFM2 antibody staining with spatial transcriptomics to correlate protein expression with local gene expression patterns

  • Implement imaging mass cytometry to analyze AIFM2 expression in relation to multiple other proteins simultaneously

  • Develop proximity ligation assays to study AIFM2 interactions with proposed partners like SIRT1 or PGC-1α

• Live-Cell Imaging Approaches:

  • Develop antibody-based biosensors for real-time monitoring of AIFM2 dynamics

  • Implement FRET-based approaches to study AIFM2 interactions in living cells

  • Use correlative light and electron microscopy to precisely locate AIFM2 at ultrastructural level

• Therapeutic Development:

  • Explore antibody-drug conjugates targeting AIFM2 in cancer cells with upregulated expression

  • Develop screening platforms using AIFM2 antibodies to identify compounds modulating its activity

  • Investigate aptamer-based approaches as alternatives to traditional antibodies