AIFM2 Antibody, Biotin conjugated

What is AIFM2 and why is it an important research target?

AIFM2, also known as Ferroptosis Suppressor Protein 1 (FSP1), functions as a NAD(P)H-dependent oxidoreductase that plays a crucial role in cellular metabolism and cell death pathways. As a key inhibitor of ferroptosis, AIFM2 catalyzes the reduction of coenzyme Q/ubiquinone-10 to ubiquinol-10, preventing lipid oxidative damage . This protein is of particular research interest due to its involvement in both apoptotic pathways and ferroptosis regulation, making it relevant for cancer research, metabolic studies, and cell death investigations .

What are the recommended storage conditions for biotin-conjugated AIFM2 antibodies?

For optimal longevity and activity, biotin-conjugated AIFM2 antibodies should be stored at -20°C for long-term storage (typically up to one year from receipt) . For short-term storage and frequent use, 4°C for up to one month is acceptable. The antibodies are typically supplied in buffer containing 50% glycerol with stabilizers such as 0.02% sodium azide and/or 0.5% BSA . Repeated freeze-thaw cycles should be avoided as they can lead to significant activity loss and protein denaturation - consider aliquoting upon first thaw if multiple uses are anticipated.

What are the validated applications for biotin-conjugated AIFM2 antibodies?

Based on validation studies, biotin-conjugated AIFM2 antibodies have been primarily optimized for:

The biotin conjugation makes these antibodies particularly valuable for multi-labeling experiments and amplification systems, though they may not be the optimal choice for all applications .

How should dilution optimization be approached for biotin-conjugated AIFM2 antibodies in ELISA?

When optimizing dilutions for biotin-conjugated AIFM2 antibodies in ELISA applications, employ a systematic titration approach:

• Begin with a broad dilution range (e.g., 1:5000, 1:10000, 1:20000, 1:40000, 1:80000)

• Use both positive controls (tissues/cells known to express AIFM2, such as heart tissue or HeLa cells)

• Include negative controls (tissues without AIFM2 expression or AIFM2-knockout samples)

• Plot signal-to-noise ratios against dilution to identify optimal concentration

• Perform secondary validation at and around the optimal dilution (e.g., if 1:40000 is optimal, test 1:30000 and 1:50000)

The exact optimal dilution will be application-specific and may need adjustment based on detection systems, sample types, and experimental conditions .

What detection systems work best with biotin-conjugated AIFM2 antibodies?

For biotin-conjugated AIFM2 antibodies, the following detection systems have demonstrated optimal performance:

When selecting a detection system, consider tissue autofluorescence, endogenous biotin levels, and experiment-specific requirements. For samples with high endogenous biotin (liver, kidney), blocking steps with free avidin/streptavidin are essential .

How does myristoylation of AIFM2 affect antibody epitope recognition, and what implications does this have for experimental design?

AIFM2 undergoes N-myristoylation at Gly-2, which mediates its recruitment to lipid droplets and plasma membrane . This post-translational modification can significantly impact epitope accessibility, particularly for antibodies targeting N-terminal regions. Researchers should consider:

• Biotin-conjugated antibodies targeting C-terminal or internal epitopes (e.g., AA 319-348) maintain consistent recognition regardless of myristoylation status

• N-terminal targeting antibodies may show differential binding between membrane-associated and cytosolic AIFM2 populations

• For comprehensive AIFM2 detection, use antibodies recognizing epitopes distant from the myristoylation site

• In subcellular localization studies, compare results using antibodies targeting different epitopes to avoid misinterpretation

This consideration is particularly important when studying AIFM2's translocation between compartments during apoptosis or stress conditions .

What are the considerations for multiplexing biotin-conjugated AIFM2 antibodies with other markers in complex experimental designs?

When designing multiplexed experiments incorporating biotin-conjugated AIFM2 antibodies:

• Biotin blocking strategy: In tissues with high endogenous biotin (liver, kidney), implement stringent avidin/biotin blocking steps before antibody application

• Selection of compatible conjugates: For other antibodies in the panel, select fluorophore or enzyme conjugates that don't utilize the biotin-streptavidin system

• Order of application: Apply biotin-conjugated AIFM2 antibodies first, followed by streptavidin detection, then proceed with subsequent antibodies

• Cross-reactivity assessment: Validate that secondary detection reagents don't cross-react with other primary antibodies in the panel

• Spectral considerations: When using fluorescent streptavidin conjugates, ensure minimal spectral overlap with other fluorophores

For co-localization studies with mitochondrial markers (particularly relevant for AIFM2), sequential rather than simultaneous application often yields cleaner results .

How do different isoforms of AIFM2 influence epitope availability, and how should researchers address this variability?

AIFM2 exists in up to two different isoforms with reported transcript lengths of 1.8 kb and 4.0 kb . This presents several methodological challenges:

• Epitope availability varies between isoforms, with some antibodies recognizing only specific variants

• The biotin-conjugated antibodies typically target conserved regions (e.g., AA 43-371) but verification is essential

• Researchers should determine which isoform(s) are relevant to their specific biological question

Recommended approach:

• Use Western blotting to confirm which isoforms are detected by your biotin-conjugated antibody

• In tissues with differential isoform expression (e.g., heart vs. liver), validate antibody performance in each tissue type

• Consider using multiple antibodies targeting different epitopes for comprehensive isoform coverage

• For quantitative studies, account for potential isoform-specific differences in signal intensity

How should researchers troubleshoot high background when using biotin-conjugated AIFM2 antibodies in immunohistochemistry?

High background is a common challenge with biotin-conjugated antibodies. A systematic troubleshooting approach includes:

For tissues with high endogenous peroxidase activity, additional peroxidase quenching (3% H₂O₂ for 10-15 minutes) may be necessary when using HRP-streptavidin detection systems .

What methodological approaches can address potential epitope masking in fixed tissues when using biotin-conjugated AIFM2 antibodies?

Epitope masking is particularly relevant for AIFM2 detection due to its complex subcellular localization. Consider these methodological approaches:

• Antigen retrieval optimization:

  • Test multiple methods: Tris-EDTA (pH 9.0) tends to work well for AIFM2

  • Compare heat-induced (pressure cooker, microwave) vs. enzymatic retrieval

  • Optimize retrieval time (10-30 minutes) for your specific tissue

• Fixation considerations:

  • Compare paraformaldehyde vs. formalin fixation results

  • For challenging samples, consider shorter fixation times

  • Test acetone or methanol fixation for certain applications

• Section thickness optimization:

  • Thinner sections (3-4 μm) generally improve antibody penetration

  • Balance section integrity with antibody accessibility

• Signal amplification:

  • Implement tyramide signal amplification compatible with biotin-conjugated antibodies

  • Consider polymer-based detection systems as alternatives

How can researchers validate the specificity of biotin-conjugated AIFM2 antibodies and distinguish between true signal and artifacts?

Rigorous validation of biotin-conjugated AIFM2 antibodies should include:

• Positive controls: Test in tissues/cells with known AIFM2 expression:

  • Heart tissue (high expression)

  • Liver and skeletal muscle (moderate expression)

  • HeLa or HepG2 cells for cell-based assays

• Negative controls:

  • Isotype-matched biotin-conjugated control antibody

  • AIFM2 knockout/knockdown samples when available

  • Pre-absorption with immunizing peptide (e.g., AA 43-371)

• Pattern validation:

  • Verify subcellular localization pattern (membrane, mitochondria, cytoplasm)

  • Compare with published localization data

  • Confirm molecular weight (41 kDa) in Western blot correlations

• Multi-antibody concordance:

  • Compare results with alternative AIFM2 antibodies targeting different epitopes

  • Verify consistent localization/expression patterns across antibodies

• Orthogonal techniques:

  • Correlate protein detection with mRNA expression

  • Confirm findings with functionally relevant assays (e.g., ferroptosis assays)

How can biotin-conjugated AIFM2 antibodies be utilized in multiplex imaging systems for studying ferroptosis mechanisms?

For researchers investigating AIFM2's role in ferroptosis regulation, multiplex approaches offer powerful insights:

• Sequential multiplex immunofluorescence:

  • Use biotin-conjugated AIFM2 antibody with streptavidin-fluorophore detection

  • After imaging, strip and reprobe with GPX4 and other ferroptosis markers

  • Digital overlay and colocalization analysis with subcellular markers

• Mass cytometry (CyTOF) integration:

  • Convert biotin-conjugated antibodies to metal-tagged probes using streptavidin-metal conjugates

  • Enables simultaneous detection of 30+ markers including AIFM2

  • Particularly valuable for analyzing heterogeneous cell populations

• Spatial transcriptomics correlation:

  • Combine AIFM2 protein detection with spatial transcriptomics

  • Correlate protein localization with gene expression patterns

  • Reveals microenvironmental influences on AIFM2 expression and function

This approach has been particularly informative in cancer research, where AIFM2/FSP1 expression correlates with resistance to ferroptosis-inducing therapies .

What considerations are important when designing proximity ligation assays (PLA) using biotin-conjugated AIFM2 antibodies?

Proximity ligation assays can reveal AIFM2 protein-protein interactions with nanometer resolution:

• Antibody compatibility assessment:

  • Biotin-conjugated AIFM2 antibody can be detected with streptavidin-oligonucleotide conjugates

  • Second antibody must target different species to avoid false positives

  • Validate epitope accessibility when both proteins are in complex

• Control design:

  • Include technical controls (single antibody controls)

  • Biological controls (interaction-deficient mutants)

  • Distance controls (proteins known to not interact with AIFM2)

• Optimization protocol:

  • Titrate both antibodies independently before combining

  • Test fixed vs. permeabilized conditions (particularly important for membrane-associated AIFM2)

  • Optimize proximity probe concentration and incubation time

• Result interpretation:

  • Quantify PLA signals per cell across multiple fields

  • Compare subcellular distribution of interaction signals

  • Correlate with functional outcomes (e.g., ferroptosis resistance)

The biotin-streptavidin system's high affinity makes it particularly suitable for PLA approaches, though careful optimization is essential.

How can biotin-conjugated AIFM2 antibodies be adapted for high-content screening applications investigating mitochondrial dynamics?

Given AIFM2's role in mitochondrial function, high-content screening offers valuable insights:

• Automated imaging workflow:

  • Biotin-conjugated AIFM2 antibody detected with streptavidin-fluorophores

  • Counterstain with mitochondrial markers (e.g., TOMM20, MitoTracker)

  • Nuclear counterstain for cell identification

  • Automated image acquisition and analysis

• Quantitative parameters:

  • AIFM2 intensity (total and mitochondrial-specific)

  • Colocalization coefficients with mitochondrial markers

  • Mitochondrial morphology metrics (size, elongation, fragmentation)

  • Translocation kinetics under stress conditions

• Experimental design considerations:

  • Plate format optimization (384-well recommended)

  • Positive controls (CCCP for mitochondrial stress)

  • Time-course imaging for dynamic processes

  • Z-stack acquisition for complete mitochondrial network analysis

This approach has proven valuable for identifying compounds that modulate AIFM2 function and mitochondrial dynamics, particularly in metabolic disease and cancer research contexts .