CIAO2A Antibody, FITC conjugated

What is CIAO2A and why is it important in cellular function?

CIAO2A (Cytosolic Iron-Sulfur Assembly Component 2A, also known as FAM96A) is a component of the cytosolic iron-sulfur protein assembly (CIA) complex with a molecular weight of approximately 18.4 kDa and a length of 160 amino acid residues. It is primarily localized in the cytoplasm and is widely expressed across many tissue types. CIAO2A functions as a critical mediator in the incorporation of iron-sulfur clusters into extramitochondrial Fe/S proteins.

The protein plays a particularly important role in cellular iron homeostasis by specifically maturing iron regulatory protein 1 (IRP1). Research has demonstrated that CIAO2A-CIA1 complexes assist specific branches of Fe/S protein assembly and intimately link this process to cellular iron regulation through both IRP1 Fe/S cluster maturation and IRP2 stabilization. Additionally, CIAO2A may contribute to chromosome segregation through the establishment of sister chromatid cohesion and potentially induce apoptosis in collaboration with APAF1.

What are the structural and functional characteristics of CIAO2A Antibody, FITC conjugated?

CIAO2A Antibody, FITC conjugated is an immunological reagent where the antibody against CIAO2A has been chemically linked to fluorescein isothiocyanate (FITC), a fluorescent dye with excitation and emission peak wavelengths of approximately 495nm and 525nm respectively. The conjugation process involves crosslinking the primary antibody with the FITC fluorophore using established protocols without significantly altering the antibody's binding specificity.

This antibody typically exists in liquid form and is preserved in solutions containing buffers such as phosphate-buffered saline (PBS) with stabilizers (often 50% glycerol) and preservatives like 0.03% Proclin 300. The FITC conjugation enables direct visualization of CIAO2A in various applications without requiring secondary antibody detection steps, which can be particularly useful in multi-labeling experiments.

What are the common applications of CIAO2A Antibody, FITC conjugated in research?

CIAO2A Antibody, FITC conjugated finds utility across multiple immunodetection techniques:

• ELISA (Enzyme-Linked Immunosorbent Assay): The primary application listed by manufacturers, used for quantitative detection of CIAO2A protein in samples.

• Immunofluorescence (IF): The FITC fluorophore enables direct visualization of CIAO2A's subcellular localization without requiring secondary antibody steps.

• Immunocytochemistry (ICC): For cellular localization studies, particularly useful when examining CIAO2A's association with other CIA complex components.

• Western Blot (WB): While not optimized for this application due to the FITC conjugation, specialized imaging systems capable of fluorescence detection can utilize this antibody for protein immunoblotting.

• Flow Cytometry: The directly conjugated format makes this antibody suitable for analyzing CIAO2A expression in cell populations or for studying its involvement in iron metabolism and apoptotic processes.

How should FITC-conjugated antibodies be stored and handled to maintain optimal performance?

Proper storage and handling of CIAO2A Antibody, FITC conjugated is essential to preserve both immunoreactivity and fluorescence intensity:

• Temperature conditions: Store at -20°C or -80°C according to manufacturer recommendations, avoiding repeated freeze-thaw cycles.

• Light protection: FITC-conjugated antibodies must be protected from continuous light exposure, which causes gradual fluorescence loss. Use amber tubes or wrap containers in aluminum foil, and work in reduced ambient lighting.

• Buffer composition: Most FITC-conjugated antibodies are supplied in PBS containing preservatives (e.g., 0.03% Proclin 300) and stabilizers (often 50% glycerol).

• Working dilutions: For immunofluorescence applications on mammalian cells, a recommended starting dilution is 1:500 in PBS containing 10% fetal bovine serum, though optimal concentration should be determined empirically for specific experimental conditions.

• Aliquoting strategy: Prepare single-use aliquots to prevent repeated freeze-thaw cycles and contamination.

How can researchers optimize dual immunolabeling protocols using CIAO2A Antibody, FITC conjugated alongside other fluorescent markers?

Successful dual immunolabeling with CIAO2A Antibody, FITC conjugated requires careful consideration of spectral properties and methodological refinements:

• Fluorophore selection: Choose secondary fluorophores with minimal spectral overlap with FITC (495nm/525nm). Recommended options include Cy3 (550nm/570nm) or Alexa Fluor 594 (590nm/617nm).

• Control implementation:

  • Perform single-color controls to establish baseline fluorescence and assess bleed-through

  • Include isotype controls conjugated to FITC to evaluate non-specific binding

  • Use unstained specimens to determine autofluorescence levels

• Antibody compatibility: When using multiple primary antibodies, ensure they originate from different host species to prevent cross-reactivity (e.g., rabbit anti-CIAO2A-FITC with mouse anti-target protein).

• Staining protocol optimization:

  • Consider sequential rather than simultaneous antibody incubation

  • Implement thorough blocking (5-10% normal serum from secondary antibody species for 30-60 minutes)

  • For CIAO2A detection, start with 1:500 dilution in PBS containing 10% FBS

• Imaging strategy: Capture each fluorophore channel separately with appropriate filter sets and combine images digitally to minimize crosstalk. Adjust acquisition parameters to account for FITC's susceptibility to photobleaching.

What methodological approaches should be employed when investigating CIAO2A's role in iron-sulfur cluster assembly pathways?

When studying CIAO2A's function in Fe-S cluster assembly using FITC-conjugated antibodies, several specialized approaches are recommended:

• Subcellular compartmentalization analysis:

  • Implement fractionation techniques to separate cytosolic, nuclear, and mitochondrial compartments

  • Use organelle-specific markers alongside CIAO2A detection to distinguish cytosolic CIA pathway from mitochondrial ISC assembly machinery

  • Consider super-resolution microscopy to visualize spatial relationships between CIAO2A and client proteins

• Protein interaction studies:

  • Examine CIAO2A's association with other CIA components (CIAO1, CIAO2B, MMS19)

  • Based on structural data, the CIA targeting complex has a potential catalytic cysteine in CIAO2B positioned ~70Å away from client protein Fe-S domains, suggesting complex structural arrangements during Fe-S transfer

  • Complement immunofluorescence with proximity ligation assays or co-immunoprecipitation to validate protein-protein interactions

• Functional Fe-S cluster assembly assessment:

  • Correlate CIAO2A localization with activity assays of Fe-S enzymes like aconitase

  • Monitor IRP1 maturation specifically, as CIAO2A has been shown to preferentially interact with this iron regulatory protein

  • Design experiments to distinguish between the functions of CIAO2A-CIA1 and CIAO2B-CIA1-MMS19 complexes, which assist different branches of Fe-S protein assembly

How can researchers verify the specificity of CIAO2A Antibody, FITC conjugated in their experimental systems?

Thorough validation of CIAO2A Antibody, FITC conjugated specificity requires a multi-faceted approach:

• Biochemical validation:

  • Perform Western blot analysis to confirm detection of a single band at the expected molecular weight of 18.4 kDa for human CIAO2A

  • Include positive controls (cells/tissues known to express CIAO2A) and negative controls (CIAO2A-depleted samples)

• Genetic verification:

  • Compare immunostaining between wild-type and CIAO2A-knockdown samples (siRNA or CRISPR-mediated)

  • Specific signals should be significantly reduced in knockdown samples

• Peptide competition assays:

  • Pre-incubate antibody with excess immunizing peptide before application

  • Specific staining should be abolished if antibody is truly specific

• Cross-reactivity assessment:

  • Verify species reactivity information from manufacturer (CIAO2A antibodies often recognize human, mouse, and rat orthologs)

  • Test reactivity across relevant species if working with non-human models

• Co-localization studies:

  • Examine co-localization with known CIAO2A interaction partners (CIAO1, components of CIA targeting complex)

  • Expected cytoplasmic localization pattern should be observed

What experimental design considerations are important when studying CIAO2A's role in iron regulation and cellular stress responses?

When investigating CIAO2A's functions in iron homeostasis and stress response pathways:

• Iron manipulation conditions:

  • Include iron chelation (e.g., deferoxamine) and iron supplementation treatments

  • Monitor CIAO2A localization, expression, and complex formation under varying iron conditions

  • Assess IRP1 activity and iron-responsive element binding under these conditions, as CIAO2A specifically matures IRP1

• Stress response analysis:

  • Examine CIAO2A dynamics during oxidative stress, which can affect Fe-S cluster integrity

  • Since CIAO2A may induce apoptosis in collaboration with APAF1, include conditions with APAF1 inhibition or knockdown

  • Monitor both IRP1 and IRP2 stabilization, as CIAO2A has been shown to impact both proteins despite IRP2 lacking an Fe-S cluster

• Temporal considerations:

  • Design time-course experiments to distinguish between immediate and adaptive responses

  • Include multiple time points before and after stress stimuli

  • Co-stain with markers for relevant stress responses (e.g., oxidative stress indicators or apoptotic markers)

• Parallel pathway analysis:

  • Compare CIA pathway components (CIAO2A, CIAO2B, CIAO1, MMS19) to understand their differential responses

  • Research has shown that CIA2B-CIA1-MMS19 and CIA2A-CIA1 assist different branches of Fe-S protein assembly

How can quantitative image analysis be optimized for CIAO2A localization studies using FITC-conjugated antibodies?

For rigorous quantification of CIAO2A localization and expression patterns:

• Image acquisition optimization:

  • Use confocal or super-resolution microscopy to minimize out-of-focus fluorescence

  • Maintain consistent acquisition parameters (exposure time, gain, laser power) across samples

  • Include fluorescence intensity calibration standards in each imaging session

  • Account for FITC's susceptibility to photobleaching by either capturing reference regions first or applying mathematical corrections

• Analysis workflow implementation:

  • Utilize software like ImageJ/Fiji or CellProfiler for quantitative analysis

  • Segment individual cells and measure parameters including integrated density, mean fluorescence intensity, and area

  • For subcellular distribution analysis, implement organelle segmentation using compartment-specific markers

• Co-localization quantification:

  • For studies examining CIAO2A's association with interaction partners (CIAO1, MMS19), use established co-localization coefficients:

    • Pearson's correlation coefficient

    • Manders' overlap coefficient

    • Object-based colocalization analysis

• Statistical considerations:

  • Analyze sufficient cell numbers (>30 per condition) across multiple biological replicates

  • Apply appropriate statistical tests based on data distribution

  • Validate microscopy-based quantification with complementary methods such as western blotting or flow cytometry