AIFM2 Antibody, FITC conjugated

What is AIFM2/FSP1 and why is it significant in research?

AIFM2 (also known as FSP1 or AMID) is a 41 kDa NAD(P)H-binding oxidoreductase belonging to the FAD-dependent oxidoreductase family. It has gained significant attention for its dual role in cell death pathways - functioning in apoptosis and as a key suppressor of ferroptosis. AIFM2/FSP1 shares sequence similarities with AIFM1 (formerly known as AIF), a mitochondrion-associated enzyme that relocates to the cell nucleus during apoptosis . Recent studies have demonstrated its importance as a parallel glutathione-independent anti-ferroptotic pathway to GPX4, making it a critical target for cancer research. The protein is primarily localized in the cell membrane, nucleus, mitochondria, and cytoplasm, with expression detected in most normal tissues .

What are the specific molecular characteristics of AIFM2/FSP1 that researchers should consider?

AIFM2/FSP1 is a 373 amino acid protein (in its canonical human form) with a molecular weight of approximately 40.5-41 kDa . It functions as an apoptosis-associated flavoprotein with a 6-hydroxy FAD cofactor . The protein has up to two known isoforms and is expressed in most normal tissues as two transcripts of 18 and 40 kb in length. When designing experiments, researchers should note that AIFM2/FSP1 has multiple subcellular localizations (cell membrane, nucleus, mitochondria, and cytoplasm), which can affect detection strategies and experimental interpretation . Its ability to relocate between cellular compartments during stress conditions is critical to its function.

What are the optimal applications for FITC-conjugated AIFM2 antibodies in research?

FITC-conjugated AIFM2 antibodies are particularly well-suited for:

For flow cytometry applications, intracellular staining protocols have been successfully validated with human cell lines including A549 cells . When used for immunofluorescence, positive staining has been confirmed in cell lines such as A549, with recommended dilutions between 1:50-1:500 .

How should researchers optimize sample preparation for FITC-conjugated AIFM2 antibody staining?

Optimal sample preparation depends on the experimental application and cellular context:

For fixed samples:

• Fixation: 4% paraformaldehyde (10-15 minutes at room temperature) preserves both protein localization and fluorescence intensity

• Permeabilization: 0.1% Triton X-100 (5-10 minutes) allows antibody access to intracellular epitopes

• Blocking: 5% normal serum (from the species of secondary antibody origin) for 30-60 minutes reduces background

For flow cytometry:

• Cell harvesting should minimize stress that might alter AIFM2 expression

• Fixation with 2% paraformaldehyde followed by permeabilization with saponin-based buffers is effective

• Single-cell suspensions should be filtered through a 40-70 μm mesh to remove aggregates

The key cellular systems validated for AIFM2 detection include HeLa cells, HepG2 cells, PC-12 cells, and A549 cells, with demonstrated reactivity in human, mouse, and rat samples .

What controls are essential when using FITC-conjugated AIFM2 antibodies?

A robust experimental design requires the following controls:

Additionally, researchers should implement technical controls such as single-color controls for compensation in multicolor flow cytometry experiments. When performing co-localization studies, fluorophore bleed-through controls are essential for accurate interpretation.

What are the critical factors for optimizing immunofluorescence protocols with FITC-conjugated AIFM2 antibodies?

Protocol optimization should focus on:

• Antibody concentration: Titration experiments starting at the manufacturer's recommended 1:50-1:500 dilution range are essential . The optimal concentration balances specific signal with minimal background.

• Incubation conditions: Test both temperature (4°C vs. room temperature) and duration (1-24 hours) to maximize signal-to-noise ratio.

• Antigen retrieval: For tissue sections, heat-induced epitope retrieval using TE buffer at pH 9.0 has been validated, though citrate buffer at pH 6.0 can serve as an alternative .

• Counterstaining: Choose nuclear counterstains that don't overlap with FITC emission spectrum (DAPI or Hoechst recommended).

• Mounting medium: Use anti-fade mounting media containing radical scavengers to preserve FITC fluorescence during imaging and storage.

• Buffer composition: PBS with 1-2% BSA at pH 7.4-8.0 (optimal for FITC) minimizes non-specific binding while maintaining FITC fluorescence efficiency.

How can researchers address weak or inconsistent FITC-AIFM2 antibody signals?

When encountering signal problems, consider the following troubleshooting approaches:

For weak signals:

• Increase antibody concentration (begin with manufacturer's recommended range 1:50-1:500 for IF/ICC)

• Optimize antigen retrieval (test both TE buffer pH 9.0 and citrate buffer pH 6.0)

• Extend incubation time (overnight at 4°C can improve signal for low-abundance proteins)

• Use signal amplification systems like tyramide signal amplification

• Check for protein degradation during sample preparation

• Ensure proper filter sets for FITC detection (excitation ~495nm, emission ~519nm)

For inconsistent results:

• Standardize fixation and permeabilization protocols across experiments

• Prepare fresh dilutions of antibody for each experiment

• Control incubation times and temperatures precisely

• Validate antibody lot-to-lot consistency

• Implement positive controls with known AIFM2 expression (HeLa, HepG2 cells)

What strategies help minimize photobleaching of FITC during imaging and analysis?

FITC is moderately susceptible to photobleaching, which can be mitigated by:

• Minimizing exposure to excitation light:

  • Reduce illumination intensity and exposure duration

  • Use neutral density filters

  • Focus on a different field than the one to be imaged

  • Employ electronic shutters to control illumination time

• Using anti-fade reagents:

  • Mount samples in commercial anti-fade media containing anti-photobleaching compounds

  • Add oxygen scavengers like glucose oxidase/catalase systems

  • Store slides at 4°C in the dark

• Optimizing image acquisition:

  • Use confocal rather than widefield microscopy when possible

  • Apply binning to reduce required exposure times

  • Capture FITC images first in multi-channel experiments

  • Utilize computational approaches like deconvolution to enhance weak signals

How can researchers resolve contradictions between AIFM2 detection methods?

When different methods yield conflicting results:

• Validate with orthogonal approaches:

  • Compare western blot (recommended dilution 1:1000-1:8000) with flow cytometry and microscopy

  • Use immunoprecipitation (0.5-4.0 μg for 1.0-3.0 mg of total protein lysate) to confirm specificity

  • Employ genetic approaches (siRNA knockdown) to confirm signal specificity

• Evaluate technical factors:

  • Different fixation methods may affect epitope accessibility

  • Various antibody clones recognize different epitopes with varying accessibility

  • Subcellular localization can affect detection (AIFM2 is found in mitochondria, cytoplasm, nucleus, and membrane)

• Consider biological variables:

  • Expression levels vary between cell types (validated in HeLa, HepG2, PC-12, A549)

  • Stress conditions may alter AIFM2 localization and detectability

  • Post-translational modifications might mask epitopes in certain contexts

How can FITC-conjugated AIFM2 antibodies be utilized to investigate ferroptosis mechanisms?

AIFM2/FSP1 has been identified as a ferroptosis suppressor, providing an opportunity to study this regulated cell death pathway:

• Co-localization studies:

  • Use FITC-AIFM2 antibodies with markers of lipid peroxidation to investigate spatial relationships

  • Examine membrane localization patterns in ferroptosis-resistant versus sensitive cells

  • Monitor AIFM2/FSP1 redistribution during ferroptosis induction

• Functional investigations:

  • Correlate AIFM2/FSP1 expression with sensitivity to ferroptosis inducers

  • Investigate the relationship between AIFM2/FSP1 levels and lipid ROS accumulation

  • Study the CoQ oxidoreductase activity of FSP1 in ferroptosis suppression

• Therapeutic relevance:

  • Screen for compounds that modulate AIFM2/FSP1 activity or localization

  • Investigate AIFM2/FSP1 upregulation as a mechanism of acquired resistance to radiotherapy

  • Explore the relationship between AIFM2/FSP1 and tumor vulnerability to ferroptosis induction

Recent publications have demonstrated AIFM2/FSP1's role as a parallel pathway to GPX4 in inhibiting ferroptosis, and upregulation of CoQ shifts ferroptosis dependence from GPX4 to FSP1 in acquired radioresistance .

What are the best practices for integrating FITC-AIFM2 antibodies into multiparameter flow cytometry panels?

When designing multicolor panels including FITC-conjugated AIFM2 antibodies:

• Panel design considerations:

  • FITC has moderate brightness and is excited by the 488nm laser

  • Compensation is critical between FITC and PE due to spectral overlap

  • Pair with spectrally distinct fluorophores like APC for co-staining

• Sample optimization:

  • Titrate FITC-AIFM2 antibody (starting at 0.25 μg per 10^6 cells)

  • Establish appropriate voltage settings using single-stained controls

  • Use fluorescence-minus-one (FMO) controls to set accurate gates

• Suggested panel for ferroptosis research:

For intracellular staining protocols, fixation and permeabilization are essential, with validated results observed in cell lines such as A549 .

How can researchers accurately quantify and interpret AIFM2 subcellular localization patterns?

Quantitative analysis of AIFM2 localization requires:

• Imaging considerations:

  • Use confocal microscopy for higher spatial resolution

  • Employ z-stacks to capture complete cellular volume

  • Include appropriate subcellular markers (mitochondria, nucleus, plasma membrane)

  • Maintain consistent acquisition settings across experimental conditions

• Quantification methods:

  • Pearson's or Mander's correlation coefficients for co-localization analysis

  • Nuclear/cytoplasmic ratio measurements

  • Membrane/cytoplasmic distribution quantification

  • Use intensity line profiles across cells to visualize distribution patterns

• Interpretation guidelines:

  • Membrane localization is critical for FSP1's anti-ferroptotic function

  • Nuclear translocation may indicate involvement in transcriptional regulation

  • Mitochondrial association suggests potential roles in mitochondrial-dependent cell death

  • Changes in localization patterns after treatments may indicate mechanism of action

What quantification approaches are most appropriate for analyzing AIFM2 expression data?

Different experimental methods require specific quantification approaches:

• Flow cytometry analysis:

  • Report median fluorescence intensity (MFI) rather than mean (less affected by outliers)

  • Use fold change in MFI relative to controls for comparisons

  • Apply appropriate statistical tests (paired t-tests for treated vs. untreated same cell line)

• Immunofluorescence quantification:

  • Measure corrected total cell fluorescence (CTCF) = Integrated Density - (Area × Mean background fluorescence)

  • Quantify subcellular distribution using nuclear/cytoplasmic ratios

  • Analyze at least 30-50 cells per condition across multiple fields

• Western blot analysis:

  • Normalize AIFM2 band intensity to loading controls (β-actin, GAPDH)

  • Use validated dilution ranges (1:1000-1:8000) for consistent results

  • Apply densitometry with appropriate background subtraction

• Statistical considerations:

  • Perform at least three independent biological replicates

  • Use non-parametric tests when normal distribution cannot be assumed

  • Apply appropriate multiple comparison corrections

How should researchers interpret changes in AIFM2/FSP1 expression in different experimental contexts?

Interpretation of AIFM2/FSP1 data requires context-specific consideration:

• Cancer research context:

  • Increased expression may indicate ferroptosis resistance mechanisms

  • Alterations in subcellular localization can suggest adaptation to stress

  • Correlate with patient outcome data when available

  • Consider the relationship with GPX4 expression (parallel ferroptosis defense)

• Cell death pathway analysis:

  • Monitor AIFM2/FSP1 in response to apoptotic vs. ferroptotic stimuli

  • Assess relationship between AIFM2/FSP1 and markers of oxidative stress

  • Evaluate changes in expression during different cell death stages

  • Consider interactions with other cell death regulators

• Therapeutic response studies:

  • AIFM2/FSP1 upregulation may indicate development of resistance

  • Changes in localization pattern can suggest mechanism of drug action

  • Alterations in post-translational modifications might affect function

  • Consider AIFM2/FSP1 as a potential biomarker for treatment response

Recent publications have demonstrated that upregulation of CoQ oxidoreductase FSP1 contributes to acquired radioresistance by shifting ferroptosis dependence from GPX4 to FSP1 .

What methodological approaches enhance reproducibility in AIFM2 antibody-based research?

To ensure reproducible results:

• Antibody validation practices:

  • Verify specificity using positive and negative controls

  • Test multiple antibody dilutions to determine optimal range

  • Document lot number and validate new lots against previous results

  • Use genetic approaches (siRNA/CRISPR) to confirm specificity

• Experimental standardization:

  • Maintain consistent fixation and permeabilization protocols

  • Use the same imaging or cytometry settings across experiments

  • Include internal standards across experimental batches

  • Document detailed protocols including timing, buffers, and temperatures

• Data management:

  • Establish clear criteria for cell/field selection prior to analysis

  • Blind analysis where possible to reduce bias

  • Use automated analysis pipelines to minimize user variability

  • Report all technical parameters in publications (antibody dilution, exposure times, etc.)

• Quality control:

  • Regularly test FITC-conjugated antibodies for fluorophore integrity

  • Monitor for consistent staining patterns in positive control samples

  • Implement periodic testing against validated standards

  • Store antibodies according to manufacturer recommendations to maintain performance

Successful applications across western blot, immunoprecipitation, immunohistochemistry, and immunofluorescence have been documented for AIFM2 antibodies, with reproducible results in human, mouse, and rat samples .