AIFM1 Antibody

What is AIFM1 and why is it important in cell death research?

AIFM1 (Apoptosis Inducing Factor Mitochondria Associated 1) is a 66.9 kDa flavoprotein with crucial roles in both mitochondrial function and caspase-independent apoptotic pathways. The protein normally resides in the mitochondrial intermembrane space where it functions as an NADH oxidase essential for oxidative phosphorylation. During apoptotic stimuli, AIFM1 translocates to the nucleus where it participates in chromatin condensation and DNA fragmentation. The protein's dual role in energy metabolism and cell death makes it a significant target in neurodegeneration, cancer, and ischemia-reperfusion injury research. AIFM1 antibodies allow researchers to track this translocation process, quantify expression levels, and understand the mechanisms that regulate this important apoptotic mediator .

What are the common aliases and orthologs of AIFM1 that researchers should be aware of?

When reviewing literature or selecting antibodies, researchers should recognize that AIFM1 appears under multiple nomenclatures across species and publications. Common aliases include: Apoptosis Inducing Factor (AIF), AIFM1, Hq (in the harlequin mouse model), AUNX1, CMT2D, and "apoptosis-inducing factor 1, mitochondrial." Based on gene homology, researchers working with animal models can find orthologs in canine, porcine, monkey, mouse, and rat systems. When designing cross-species experiments or comparing results from different model organisms, understanding these alternative designations is crucial for comprehensive literature searches and proper antibody selection .

What is the basic structural and functional organization of the AIFM1 protein?

AIFM1 exhibits a complex multi-domain architecture that dictates its diverse functions in cellular homeostasis and death pathways. The mature 62 kDa protein contains:

• N-terminal mitochondrial localization sequence (MLS, residues 1-35)

• FAD-binding domain (residues 122-262 and 400-477)

• NADH-binding domain (residues 263-399)

• C-terminal domain containing a nuclear localization sequence (NLS)

• Reactive cysteine residues (notably Cys-255 and Cys-440)

During apoptotic signaling, proteolytic cleavage by calpains or cathepsins removes the N-terminal MLS, generating a truncated AIF protein (tAIF) of approximately 57 kDa that can translocate to the nucleus. Phosphorylation at specific residues, particularly Ser-116, regulates this translocation process. When selecting antibodies, researchers should consider which domain they wish to target based on their experimental objectives—whether tracking full-length protein, cleaved forms, or specific post-translational modifications .

What criteria should researchers use when selecting an AIFM1 antibody for specific applications?

Researchers should evaluate multiple parameters when selecting an AIFM1 antibody:

The search results indicate over 550 AIFM1 antibodies available commercially with varying applications and specificities. Researchers investigating mitochondrial-to-nuclear translocation events should prioritize antibodies validated for both immunocytochemistry and subcellular fractionation approaches .

How can researchers validate the specificity of an AIFM1 antibody in their experimental system?

• Positive and negative controls:

  • Positive: Tissue/cells known to express AIFM1 (widespread expression, particularly high in brain, heart, muscle)

  • Negative: AIFM1 knockout cells/tissues or siRNA knockdown samples

• Molecular weight verification: Confirm detection at expected sizes (full-length ~67 kDa, cleaved form ~57 kDa) in Western blot

• Peptide competition assay: Pre-incubating antibody with immunizing peptide should abolish specific signal

• Multiple antibody concordance: Results should be reproducible with antibodies targeting different epitopes

• Subcellular localization pattern: In healthy cells, AIFM1 should localize predominantly to mitochondria; during apoptosis, nuclear translocation should be observable

• Stimulation response: Treatment with known inducers of AIFM1 translocation (e.g., PARP-activating agents) should produce expected changes in localization and/or processing

Careful validation eliminates the risk of non-specific binding that could lead to misinterpretation of experimental results, especially in complex disease models .

What are the optimal protocols for using AIFM1 antibodies in Western blotting?

Western blotting for AIFM1 requires careful optimization to detect both full-length and processed forms of the protein. A recommended protocol includes:

• Sample preparation:

  • For total AIFM1: Standard RIPA buffer with protease inhibitors

  • For distinguishing subcellular pools: Perform fractionation to separate mitochondrial, cytosolic, and nuclear fractions

  • Use fresh samples when possible or store at -80°C with protease inhibitors

• Gel separation:

  • 10-12% SDS-PAGE for optimal resolution of 57-67 kDa range

  • Include positive control lysates (e.g., HeLa cells treated with staurosporine)

  • Load 20-50 μg total protein per lane

• Transfer and blocking:

  • PVDF membrane (rather than nitrocellulose) works optimally for AIFM1

  • Transfer at 100V for 60-90 minutes in cold room

  • Block with 5% non-fat milk or BSA in TBST for 1 hour

• Antibody incubation:

  • Primary: 1:500-1:2000 dilution (optimize for each antibody), overnight at 4°C

  • Secondary: HRP-conjugated, 1:5000-1:10000, 1 hour at room temperature

  • Wash thoroughly (3-5 times, 5-10 minutes each) between incubations

• Detection considerations:

  • Enhanced chemiluminescence (ECL) with film or digital imaging

  • For quantification of both full-length and cleaved forms, ensure linear range detection

When analyzing results, researchers should note that AIFM1 antibodies may detect multiple bands: the full-length ~67 kDa form, the processed ~57 kDa form during apoptosis, and occasionally other processed fragments depending on the cell type and apoptotic stimulus .

How should researchers optimize immunocytochemistry protocols for studying AIFM1 translocation?

Studying AIFM1 translocation from mitochondria to nucleus requires careful immunocytochemistry protocol optimization:

• Fixation options:

  • 4% paraformaldehyde (10 minutes at room temperature) preserves most epitopes

  • For some antibodies, methanol fixation (-20°C, 10 minutes) may better preserve mitochondrial structures

  • Avoid extended fixation which can mask epitopes

• Permeabilization:

  • 0.1-0.2% Triton X-100 (10 minutes) or 0.1% saponin for gentler permeabilization

  • For phospho-specific antibodies, include phosphatase inhibitors in all buffers

• Blocking and antibody incubations:

  • Block with 5-10% normal serum from secondary antibody species

  • Primary antibody dilutions typically 1:100-1:500, overnight at 4°C

  • Secondary antibody dilutions 1:200-1:1000, 1 hour at room temperature

• Co-staining recommendations:

  • Mitochondrial marker (e.g., MitoTracker, Tom20, or COXIV)

  • Nuclear counterstain (e.g., DAPI or Hoechst)

  • For advanced studies, combine with other apoptotic markers

• Imaging considerations:

  • Confocal microscopy provides optimal resolution for colocalization studies

  • Z-stack acquisition for 3D visualization of translocation events

  • Time-lapse imaging for monitoring translocation kinetics in live cells

Researchers should validate translocation by quantifying nuclear/mitochondrial AIFM1 ratios across multiple cells. Positive controls (e.g., cells treated with H2O2, staurosporine, or MNNG) should be included to confirm antibody ability to detect translocated AIFM1 .

What are the most informative flow cytometry approaches for studying AIFM1 in apoptotic pathways?

Flow cytometry provides quantitative analysis of AIFM1 dynamics across cell populations during apoptosis. Optimal approaches include:

• Intracellular AIFM1 staining protocol:

  • Fix cells in 2% paraformaldehyde (15 minutes, room temperature)

  • Permeabilize with 0.1% saponin or commercial permeabilization buffer

  • Block with 3% BSA in PBS (30 minutes)

  • Incubate with primary AIFM1 antibody (1:100-1:200, 60 minutes)

  • Wash 3× with PBS/0.1% saponin

  • Incubate with fluorophore-conjugated secondary antibody (1:500, 45 minutes)

  • Include unstained, secondary-only, and isotype controls

• Multi-parametric analysis combinations:

  • AIFM1 + mitochondrial membrane potential dye (TMRE or JC-1)

  • AIFM1 + cell death markers (Annexin V, PI)

  • AIFM1 + DNA content (cell cycle analysis)

  • AIFM1 + caspase activation markers

• Time course studies:

  • Collect samples at multiple timepoints after apoptotic stimulation

  • Track changes in AIFM1 subcellular distribution and processing

• Gating strategy:

  • Exclude debris and doublets

  • Gate on specific cell populations of interest

  • Create bivariate plots of AIFM1 vs. other apoptotic markers

The key advantage of flow cytometry is the ability to correlate AIFM1 status with other apoptotic parameters at the single-cell level, revealing population heterogeneity and temporal dynamics that might be missed in bulk assays .

What are the common technical challenges when working with AIFM1 antibodies in Western blotting?

Researchers frequently encounter specific challenges when using AIFM1 antibodies in Western blotting:

When AIFM1 antibody performance is suboptimal, researchers should consider that some epitopes might be masked by fixation methods, protein-protein interactions, or conformational changes during apoptosis. Testing multiple antibodies targeting different regions of the protein can help resolve these issues .

How can researchers address non-specific binding issues with AIFM1 antibodies in tissue immunohistochemistry?

Non-specific binding in tissue immunohistochemistry with AIFM1 antibodies can complicate interpretation of results, especially in tissues with high endogenous peroxidase activity or autofluorescence. To address these challenges:

• Enhanced blocking protocols:

  • Extend blocking time to 2 hours with 10% normal serum

  • Add 0.1-0.3% Triton X-100 to blocking buffer

  • Include 0.1% BSA and 0.05% Tween-20 in all antibody diluents

  • For highly problematic tissues, use avidin/biotin blocking kit

• Antigen retrieval optimization:

  • Test multiple methods: heat-induced (citrate buffer pH 6.0 vs. EDTA buffer pH 9.0)

  • Enzymatic retrieval (proteinase K) may be more effective for some fixed tissues

  • Careful timing is critical—excessive retrieval can increase non-specific binding

• Controls for validation:

  • Pre-absorption with immunizing peptide

  • Omission of primary antibody

  • Gradient of primary antibody concentrations

  • Tissue from AIFM1 knockout models (when available)

  • Parallel staining with alternative antibody clones

• Signal enhancement with minimal background:

  • Tyramide signal amplification systems for low-abundance detection

  • Polymer-based detection systems instead of ABC method

  • Enhanced washing protocols (longer washes, higher salt concentration)

Researchers should be particularly cautious when examining tissues with high endogenous AIFM1 expression (brain, heart, liver) where distinguishing specific from non-specific signal can be challenging. Progressive dilution series of the primary antibody can help identify the optimal concentration that maximizes signal-to-noise ratio .

How can phospho-specific AIFM1 antibodies be utilized to study regulatory mechanisms of AIFM1 translocation?

Phosphorylation of AIFM1, particularly at Ser-116, plays a critical role in regulating its apoptotic function and translocation. Phospho-specific antibodies provide unique insights into these regulatory mechanisms:

• Key phosphorylation sites and their significance:

  • Ser-116: Phosphorylation by GSK3β inhibits AIFM1 release and nuclear translocation

  • Other sites (Thr-263, Ser-479) may influence protein-protein interactions and activity

• Experimental approaches with phospho-AIFM1 antibodies:

  • Western blotting: Monitor phosphorylation status during apoptotic progression

  • Immunoprecipitation: Identify binding partners specific to phosphorylated forms

  • Immunofluorescence: Track subcellular distribution of phosphorylated AIFM1

  • FRET/BRET assays: Study dynamic conformational changes after phosphorylation

• Methodological considerations:

  • Include phosphatase inhibitors in all buffers

  • Use phos-tag gels for enhanced separation of phosphorylated forms

  • Validate phospho-antibody specificity with phosphatase treatment

  • Compare phospho-null (S116A) and phospho-mimetic (S116D) AIFM1 mutants

• Applications in signaling pathway analysis:

  • Screen kinase inhibitors for effects on AIFM1 phosphorylation status

  • Correlate phosphorylation with apoptotic resistance in cancer models

  • Analyze phosphorylation changes in response to oxidative stress or DNA damage

Phospho-specific antibodies have revealed that AIFM1 phosphorylation status can differ between tumor types and correlate with treatment resistance. For example, increased Ser-116 phosphorylation has been observed in certain chemoresistant cancer cells, suggesting a mechanism by which these cells evade AIF-mediated cell death .

What are the considerations for using AIFM1 antibodies in chromatin immunoprecipitation (ChIP) experiments?

While AIFM1 is not a classical DNA-binding transcription factor, it associates with chromatin during apoptosis and may interact with specific genomic regions. ChIP experiments with AIFM1 antibodies require special considerations:

• Crosslinking optimization:

  • Standard 1% formaldehyde (10 minutes) may be insufficient

  • Test dual crosslinking with 1 mM DSG (disuccinimidyl glutarate) followed by formaldehyde

  • For detecting weaker interactions, extend crosslinking time (15-20 minutes)

• Sonication parameters:

  • AIFM1-DNA complexes may require different fragmentation conditions

  • Aim for slightly larger fragments (300-500 bp) than standard ChIP

  • Verify fragmentation efficiency by agarose gel electrophoresis

• Antibody selection criteria:

  • Use antibodies specifically validated for ChIP applications

  • Target C-terminal regions more likely to be accessible in DNA-bound forms

  • Avoid phospho-specific antibodies unless studying specific regulatory events

• Controls and validation:

  • Include IgG negative control and positive control for known DNA-binding protein

  • Perform parallel ChIP with different AIFM1 antibodies targeting distinct epitopes

  • Validate enrichment by qPCR before proceeding to sequencing

  • Include input normalization and spike-in controls for quantitative analysis

• Data analysis considerations:

  • Compare AIFM1 binding patterns before and after apoptotic stimulation

  • Correlate with chromatin accessibility data (ATAC-seq) and histone modifications

  • Search for co-occurrence with known partners (CypA, H2AX, EndoG)

Researchers should note that AIFM1-associated chromatin regions may not display the sharp peaks typical of sequence-specific transcription factors, but rather broader regions of enrichment similar to chromatin modifiers. This pattern reflects AIFM1's role in higher-order chromatin reorganization during apoptosis rather than sequence-specific binding .

How can quantitative proteomics approaches be combined with AIFM1 antibodies to identify novel interaction partners?

Combining immunoprecipitation of AIFM1 with mass spectrometry-based proteomics provides powerful insights into its protein interaction network across different cellular states. Advanced methodological approaches include:

• Optimized immunoprecipitation strategies:

  • Native IP: Preserves weaker interactions and complexes

  • Crosslinking IP: Captures transient interactions

  • Proximity labeling: BioID or APEX2 fusions to capture neighborhood proteins

  • Comparison of different antibodies targeting distinct AIFM1 domains

• Specialized lysis conditions:

  • Mitochondrial vs. nuclear fractions to distinguish compartment-specific interactomes

  • Healthy vs. apoptotic cells to identify stimulus-dependent interactions

  • Detergent optimization to preserve membrane-associated complexes

• Mass spectrometry preparation:

  • On-bead digestion minimizes contamination and sample loss

  • FASP (Filter-Aided Sample Preparation) for enhanced peptide recovery

  • TMT or iTRAQ labeling for multiplexed quantitative comparison

  • Sequential elution for differentiation of specific vs. non-specific binders

• Bioinformatic analysis pipeline:

  • SAINT or CRAPome filtering to remove common contaminants

  • GO/KEGG pathway enrichment of interacting proteins

  • Protein interaction network visualization with Cytoscape

  • Domain enrichment analysis to identify common structural features

• Validation of novel interactions:

  • Reciprocal co-IP with antibodies against newly identified partners

  • Proximity ligation assay (PLA) to confirm interactions in situ

  • FRET/BRET assays for dynamic interaction analysis

  • Functional studies with siRNA knockdown of interaction partners

This approach has revealed previously unappreciated AIFM1 interactions with proteins involved in RNA metabolism, redox homeostasis, and DNA damage response pathways. Researchers should pay particular attention to interaction differences between full-length and cleaved AIFM1 forms, as these may reveal mechanisms controlling the switch between its metabolic and apoptotic functions .

What emerging technologies are enhancing AIFM1 antibody-based research?

Recent technological advances are transforming how researchers utilize AIFM1 antibodies in multiple research contexts:

• Advanced imaging applications:

  • Super-resolution microscopy (STED, STORM) for nanoscale AIFM1 localization

  • Lattice light-sheet microscopy for long-term live-cell AIFM1 dynamics

  • Correlative light-electron microscopy to connect AIFM1 signal with ultrastructure

  • Light-inducible protein technologies to trigger AIFM1 translocation

• Single-cell approaches:

  • CyTOF/mass cytometry with metal-conjugated AIFM1 antibodies

  • Microfluidic single-cell Western blotting for heterogeneity analysis

  • Spatial transcriptomics combined with AIFM1 immunofluorescence

  • Digital spatial profiling for tissue microenvironment analysis

• Nanoantibody and recombinant antibody technologies:

  • AIFM1-specific nanobodies for live-cell tracking

  • Bispecific antibodies linking AIFM1 to specific cellular compartments

  • Degradation-targeting chimeric antibodies to modulate AIFM1 levels

  • Intrabodies expressed from genetic constructs for in vivo studies

• High-throughput screening applications:

  • AIFM1 translocation-based drug screening platforms

  • CRISPR screens for genes affecting AIFM1 localization

  • Automated imaging systems for population-level dynamics

  • Machine learning classification of AIFM1 translocation patterns

These emerging approaches are enabling researchers to address previously intractable questions about the spatial and temporal dynamics of AIFM1 function across diverse physiological and pathological contexts .

What considerations are important when interpreting AIFM1 antibody data in the context of neurological disease research?

AIFM1 has been implicated in various neurological disorders, from acute conditions like stroke to neurodegenerative diseases such as Alzheimer's and Parkinson's. When interpreting AIFM1 antibody data in neurological disease contexts, researchers should consider:

• Disease-specific AIFM1 modifications:

  • Alternative splicing variants may be tissue or disease-specific

  • Post-translational modifications can differ between healthy and diseased states

  • Proteolytic processing patterns may vary by pathological condition

  • Antibody epitope accessibility may change in protein aggregates

• Cell type heterogeneity considerations:

  • Neurons, astrocytes, microglia show different AIFM1 expression patterns

  • Cell type-specific responses to injury may affect AIFM1 dynamics

  • Mixed cell populations may obscure cell type-specific changes

  • Single-cell approaches or cell sorting may be necessary for clarity

• Technical validation for neurological samples:

  • Fixation artifacts are common in brain tissue

  • Autofluorescence is particularly problematic in aged brain samples

  • Protein extraction from neural tissues may require specialized buffers

  • Post-mortem interval affects protein integrity and modified forms

• Data interpretation challenges:

  • Distinguishing causative vs. consequential AIFM1 changes

  • Correlation with disease progression markers

  • Integration with genetic data (e.g., AIFM1 mutations in CMTD)

  • Translation between animal models and human pathology

• Therapeutic targeting considerations:

  • AIFM1 inhibitors vs. activators may be context-dependent

  • Timing of intervention relative to disease stage

  • Cell type-specific targeting approaches

  • Biomarker potential of different AIFM1 forms

In neurological disease research, AIFM1 antibody studies should ideally be combined with functional readouts of mitochondrial integrity, reactive oxygen species production, and cell death pathway activation to provide a comprehensive understanding of AIFM1's role in disease pathogenesis and potential as a therapeutic target .