IAN2 Antibody

What is IAN2 and why is it significant in research?

IAN2 (GTPase IMAP family member 6) is a protein with a canonical amino acid length of 292 residues and a molecular mass of 32.9 kilodaltons in humans. It belongs to the AIG1/Toc34/Toc159-like paraseptin GTPase protein family and is primarily localized in the cytoplasm. Its significance stems from its wide expression across multiple tissue types and potential roles in immune regulation and cellular processes. Understanding IAN2 function contributes to broader research on cellular signaling and immune system regulation .

What experimental applications are most suitable for IAN2 antibodies?

IAN2 antibodies are primarily utilized in Western Blot (WB) and ELISA applications. These techniques allow researchers to detect and quantify IAN2 protein in biological samples, respectively. The antibody's specificity for Arabidopsis also makes it valuable for plant biology research examining conserved immune pathways . When designing experiments, researchers should consider the specific isoform of interest, as three distinct isoforms have been identified that may exhibit different expression patterns or functional properties.

How do I select the appropriate IAN2 antibody for my research?

Selection should be based on multiple factors including:

• Target specificity - confirm the antibody recognizes your specific IAN2 isoform of interest

• Host species - consider potential cross-reactivity issues

• Application compatibility - verify validation for your specific application (WB, ELISA, etc.)

• Clonality - polyclonal antibodies offer broader epitope recognition while monoclonals provide higher specificity

• Validation data - review available data demonstrating specificity in your experimental system

For Arabidopsis research specifically, commercially available antibodies from suppliers like CUSABIO and MyBioSource have been developed with appropriate reactivity profiles .

What controls should be included when using IAN2 antibodies?

Robust experimental design with IAN2 antibodies requires multiple controls:

• Positive control: Sample known to express IAN2 (based on tissue expression data)

• Negative control: Sample known to lack IAN2 expression

• Isotype control: Non-specific antibody of the same isotype and host species

• Blocking peptide control: Pre-incubation of antibody with target antigen

• Loading/housekeeping controls: For normalization in quantitative applications

These controls are critical for validating antibody specificity and distinguishing true signal from background. For Western blots specifically, molecular weight markers help confirm the expected 32.9 kDa band for canonical IAN2, though variant isoforms may appear at different molecular weights .

How can I optimize Western blot protocols for IAN2 detection?

Optimizing Western blot protocols for IAN2 detection requires attention to several parameters:

• Sample preparation: Complete lysis with appropriate buffers containing protease inhibitors

• Protein loading: 20-50 µg total protein per lane typically provides adequate detection

• Separation: 10-12% acrylamide gels effectively resolve the 32.9 kDa IAN2 protein

• Transfer conditions: Semi-dry transfer at 15V for 30 minutes or wet transfer at 100V for 1 hour

• Blocking: 5% non-fat dry milk in TBST for 1 hour at room temperature

• Primary antibody: Dilution ranges typically between 1:500-1:2000, incubate overnight at 4°C

• Secondary antibody: HRP-conjugated anti-host IgG at 1:5000-1:10000 for 1 hour

• Detection: Enhanced chemiluminescence with exposure times optimized based on signal strength

For plant samples, additional steps to remove interfering compounds may be necessary, as commercial IAN2 antibodies show reactivity with Arabidopsis .

What are the key considerations for ELISA-based quantification of IAN2?

ELISA-based quantification of IAN2 involves several critical considerations:

• Antibody pairing: For sandwich ELISA, use capture and detection antibodies recognizing different epitopes

• Standard curve: Generate using recombinant IAN2 protein at 0-1000 ng/mL

• Sample dilution: Perform serial dilutions to ensure readings fall within the linear range

• Coating concentration: Typically 1-5 µg/mL of capture antibody

• Blocking: 1-3% BSA in PBS to minimize background

• Sample volume: 100 µL per well standardized across plate

• Incubation times: 1-2 hours at room temperature for each step

• Washing: At least 3-5 washes with PBS-T between steps

• Detection system: HRP-conjugated secondary antibody with TMB substrate

• Data analysis: Four-parameter logistic regression for standard curve fitting

The current commercial IAN2 antibodies have been validated for ELISA applications, particularly with plant samples .

How can I investigate IAN2 protein-protein interactions in cellular systems?

Investigating IAN2 protein-protein interactions requires specialized approaches:

• Co-immunoprecipitation (Co-IP): Use IAN2 antibodies to pull down protein complexes

  • Crosslinking may be necessary for transient interactions

  • Use gentle lysis conditions to preserve native complexes

  • Include RNase/DNase treatment to eliminate nucleic acid-mediated associations

• Proximity ligation assay (PLA): Visualize interactions in situ

  • Requires antibodies from different host species

  • Provides spatial information about interaction sites within cells

• FRET/BRET analysis: For live-cell interaction studies

  • Requires fusion proteins with appropriate fluorophores/luciferase

  • Allows real-time monitoring of dynamic interactions

• Yeast two-hybrid screening: For novel interaction partner discovery

  • Use IAN2 as bait to screen cDNA libraries

  • Validate hits with reciprocal Co-IP experiments

As a member of the GTPase family, IAN2 likely engages in numerous protein interactions that regulate its function and localization within the cell .

What approaches can address antibody cross-reactivity issues in IAN2 research?

Addressing cross-reactivity issues requires systematic troubleshooting:

• Epitope mapping: Identify the specific sequence recognized by the antibody

  • Peptide arrays can pinpoint exact binding regions

  • Knowledge of epitope helps predict potential cross-reactivity

• Knockout/knockdown validation: Test antibody in systems lacking IAN2

  • CRISPR-Cas9 knockout cells provide definitive negative controls

  • siRNA knockdown samples should show reduced signal intensity

• Pre-absorption: Incubate antibody with purified antigen before application

  • Specific signal should disappear while cross-reactive bands remain

• Alternative antibody clones: Test multiple antibodies targeting different epitopes

  • Consistent detection across antibodies increases confidence

• Mass spectrometry validation: Confirm identity of detected proteins

  • Immunoprecipitate the target and perform MS analysis

  • Verify presence of IAN2 peptides in the detected band

This systematic approach is particularly important since IAN2 belongs to a family of related proteins with potential sequence homology .

How can I study IAN2 localization and trafficking in living cells?

Studying IAN2 localization and trafficking requires dynamic imaging approaches:

• Immunofluorescence microscopy:

  • Fix cells with 4% paraformaldehyde (10 min, RT)

  • Permeabilize with 0.1% Triton X-100 (5 min, RT)

  • Block with 5% normal serum (1 hour, RT)

  • Incubate with anti-IAN2 primary antibody (1:100-1:500, overnight, 4°C)

  • Apply fluorophore-conjugated secondary antibody (1:500, 1 hour, RT)

  • Counterstain nuclei with DAPI (1:1000, 5 min, RT)

• Live-cell imaging with fluorescent fusion proteins:

  • Generate IAN2-GFP expression constructs

  • Transfect cells using appropriate method (lipofection, electroporation)

  • Image using confocal microscopy with environmental control

  • Photobleaching techniques (FRAP/FLIP) can assess protein mobility

• Subcellular fractionation with Western blot:

  • Separate nuclear, cytoplasmic, membrane, and organelle fractions

  • Confirm fraction purity with compartment-specific markers

  • Probe fractions with anti-IAN2 antibody

Based on current knowledge, IAN2 predominantly localizes to the cytoplasm, though specific subcompartment localization may vary by cell type or condition .

How does IAN2 expression compare across different tissues and disease states?

IAN2 expression analysis requires careful experimental design and data interpretation:

When interpreting expression data:

• Normalize to appropriate housekeeping genes/proteins

• Consider isoform-specific expression patterns

• Compare multiple detection methods for confirmation

• Account for post-translational modifications that may affect antibody recognition

• Validate findings across multiple biological replicates

IAN2 is widely expressed across many tissue types, making comparative analysis valuable for understanding tissue-specific functions .

What methodological approaches help distinguish between IAN2 isoforms?

Distinguishing between the three reported IAN2 isoforms requires targeted strategies:

• Isoform-specific PCR primers:

  • Design primers spanning unique exon junctions

  • Optimize annealing temperatures for specificity

  • Validate with known isoform-expressing controls

• Custom antibodies:

  • Generate antibodies against isoform-specific regions

  • Validate using overexpression systems of each isoform

• Mass spectrometry:

  • Identify unique peptide sequences from each isoform

  • Develop selected reaction monitoring (SRM) assays

• 2D gel electrophoresis:

  • Separate isoforms by both pI and molecular weight

  • Confirm identity via Western blot or MS analysis

• Targeted RNA-seq analysis:

  • Quantify isoform-specific exon usage

  • Calculate relative abundance of each transcript variant

This comprehensive approach allows researchers to determine which specific isoform(s) are relevant to their biological system of interest .

What is known about the functional role of IAN2 compared to other GTPase family members?

Understanding IAN2's functional role requires comparison with related proteins:

• Comparative biochemical analysis:

  • GTPase activity assays (colorimetric phosphate release, fluorescent GTP analogs)

  • Nucleotide binding preferences (GTP vs. GDP affinity)

  • Structural analysis (X-ray crystallography, cryo-EM)

• Evolutionary conservation studies:

  • Phylogenetic analysis across species

  • Identification of conserved functional domains

  • Cross-species rescue experiments

• Interactome comparison:

  • Mass spectrometry-based interactor identification

  • Yeast two-hybrid screening against cDNA libraries

  • Bioinformatic prediction of interaction networks

As a member of the AIG1/Toc34/Toc159-like paraseptin GTPase family, IAN2 likely shares structural features with related proteins while potentially having divergent functions in specific cellular contexts .

What storage and handling conditions maximize IAN2 antibody stability and performance?

Optimal handling and storage practices for IAN2 antibodies include:

• Storage temperature: Store at 2-8°C for short-term (1-2 weeks) or aliquot and store at -20°C for long-term

• Freeze-thaw cycles: Minimize to ≤5 cycles by preparing small working aliquots

• Buffer composition: Standard antibody solutions contain 0.1% sodium azide and protein stabilizers

• Working dilution preparation: Dilute in appropriate buffer immediately before use

• Stability indicators: Monitor for signs of precipitation, color change, or reduced activity

• Temperature control: Maintain cold chain during shipping and handling

• Documentation: Record lot numbers, receipt dates, and performance characteristics

Proper storage and handling are critical for maintaining antibody specificity and sensitivity over time .

How can I validate the specificity of an IAN2 antibody for my experimental system?

Comprehensive antibody validation strategies include:

• Genetic controls:

  • Test in IAN2 knockout/knockdown systems

  • Compare with IAN2 overexpression samples

• Antigen competition:

  • Pre-incubate antibody with excess purified antigen

  • Specific signal should be eliminated or significantly reduced

• Independent detection methods:

  • Compare results with alternative antibodies targeting different epitopes

  • Correlate with mRNA expression data

• Cross-reactivity assessment:

  • Test against closely related family members

  • Examine reactivity in species expected to lack homologs

• Application-specific validation:

  • For Western blot: Confirm expected molecular weight (32.9 kDa for canonical form)

  • For ELISA: Establish dose-response relationship and specificity

This multi-faceted validation approach ensures experimental results are attributable to specific IAN2 detection rather than artifacts or cross-reactivity .

What methodological modifications are necessary when studying IAN2 in different model systems?

Adapting IAN2 research methods across model systems requires specific considerations:

• Plant systems (e.g., Arabidopsis):

  • Modified extraction buffers to handle plant-specific compounds

  • Increased detergent concentrations may be needed for membrane extraction

  • Commercial antibodies from CUSABIO and MyBioSource show reactivity

• Mammalian cell culture:

  • Standard RIPA or NP-40 buffers are typically sufficient

  • Cell-type specific optimization of lysate preparation may be necessary

• Tissue specimens:

  • Optimized fixation protocols for immunohistochemistry (4% PFA, 10% NBF)

  • Antigen retrieval methods may need empirical determination

  • Consider tissue-specific expression levels for loading adjustments

• Bacteria/yeast expression systems:

  • Codon optimization for heterologous expression

  • Inclusion body solubilization protocols for purification

  • Tag selection to minimize interference with protein function

These methodological adaptations ensure comparable and reliable results across diverse experimental models .