IRF7 Antibody, FITC conjugated

What is the functional significance of IRF7 and why would researchers use a FITC-conjugated antibody for its detection?

IRF7 functions as a master transcriptional regulator of type I interferons, playing a crucial role in antiviral immune responses. Located primarily in the nucleus, IRF7 becomes activated in response to viral infections and is essential for the transcriptional activation of interferon-α and interferon-β genes . This nuclear localization enables IRF7 to quickly respond to viral signals and initiate immune responses, enhancing the host's ability to combat infections. IRF7 also interacts with other proteins in the interferon signaling pathway, including IRF-3, which further underscores its importance in regulating immune responses .

Researchers utilize FITC-conjugated IRF7 antibodies because the fluorescent tag enables direct visualization of IRF7 protein in cellular contexts without requiring secondary antibody steps. This is particularly valuable for immunofluorescence studies, flow cytometry, and microscopy applications where researchers need to track IRF7 nuclear translocation following viral infection or other immune stimuli. The bright green fluorescence of FITC provides excellent signal-to-noise ratio for detecting changes in IRF7 expression levels and subcellular localization .

What are the key structural domains of IRF7 that might impact antibody binding and experimental design?

IRF7 possesses a complex domain structure that researchers must consider when designing experiments with antibodies. The protein contains several functionally distinct regions:

• DNA Binding Domain (DBD): Located at amino acids 1-150 in the N-terminal region, present in all IRF family members

• Constitutive Activation Domain (CAD): Found between amino acids 151-246, maintains the activity of IRF7

• Virus Activated Domain (VAD): Located between amino acids 278-305, essential for IRF7 activation in response to viral infection

• Inhibitory Domain (ID): Located between amino acids 341-467, interferes with the transactivation function of IRF7; contains a nuclear export signal (NES)

• Signal Response Domain (SRD): Located at the C-terminal end between amino acids 468-503, mediates IRF7 dimerization and contains a serine-rich region critical for phosphorylation-dependent activation

These structural features are important considerations when selecting antibodies for specific applications. For instance, antibodies targeting the SRD region might be ideal for studying phosphorylation-dependent activation, while those targeting the DBD might interfere with DNA binding in certain assays. Epitope mapping is therefore crucial when selecting an antibody for a specific research application .

How do I properly store and handle IRF7 Antibody, FITC conjugated to maintain its functionality?

Proper storage and handling of FITC-conjugated IRF7 antibodies is essential to preserve functionality and prevent signal degradation. Upon receipt, store the antibody at -20°C or -80°C to maintain stability . Avoid repeated freeze-thaw cycles as these can significantly reduce antibody activity and fluorophore brightness .

When working with the antibody:

• Aliquot the stock solution into smaller volumes upon first thaw to minimize freeze-thaw cycles

• Protect from light during all handling procedures, as FITC is photosensitive and will photobleach with extended light exposure

• Maintain proper buffer conditions (typically the antibody is stored in 50% glycerol, 0.01M PBS, pH 7.4 with 0.03% Proclin 300 as preservative)

• Allow the antibody to reach room temperature before opening the vial to prevent condensation that could introduce contamination

• Use sterile technique when handling to prevent microbial contamination

For long-term storage, keep the antibody in the dark at the recommended temperature. When diluting for experiments, use fresh buffer systems and consider adding protein carriers (such as BSA) to prevent non-specific binding and surface adsorption issues that can reduce effective concentration .

What are the recommended applications for IRF7 Antibody, FITC conjugated, and how should protocols be optimized?

IRF7 antibodies with FITC conjugation are suitable for multiple applications with different optimization requirements:

Flow Cytometry:

• Optimal dilution: Usually 1:50-1:200, requiring titration for each lot

• Cell preparation: Fixation with 4% paraformaldehyde followed by permeabilization with 0.1% Triton X-100 or methanol for nuclear antigen access

• Controls: Include isotype control (FITC-conjugated IgG or IgG2a) to determine background fluorescence

Immunofluorescence Microscopy:

• Optimal dilution: Typically 1:100-1:500 for tissue sections or cultured cells

• Sample preparation: Proper fixation (paraformaldehyde) and permeabilization is critical for nuclear antigens like IRF7

• Counterstaining: Use DAPI for nuclear visualization to confirm IRF7 nuclear translocation

ELISA:

• While both monoclonal and polyclonal IRF7 antibodies are applicable for ELISA , FITC conjugates are less commonly used for this purpose unless developing a fluorescence-based ELISA

Western Blotting:

• Although FITC-conjugated antibodies can be used for direct fluorescence detection on blots, they typically provide lower sensitivity than HRP-conjugated alternatives

Protocol optimization should include:

• Titration of antibody concentration for each application

• Determination of optimal fixation and permeabilization conditions

• Inclusion of appropriate blocking reagents to minimize non-specific binding

• Validation of specificity using positive and negative controls

What are the species reactivity considerations when using IRF7 antibodies in different research models?

Species reactivity is a critical consideration that directly impacts experimental design and interpretation. Different IRF7 antibodies show varying reactivity patterns:

When designing experiments with multiple species, consider:

• Sequence homology: Human IRF7 shares approximately 70% amino acid identity with mouse IRF7, but critical functional domains are more highly conserved

• Epitope accessibility: Even conserved regions may show differential antibody accessibility due to species-specific post-translational modifications

• Cross-validation: For novel models, validate antibody binding with positive and negative controls from the species of interest

• Alternative isoforms: IRF7 has multiple isoforms that might be differentially expressed across species

Researchers should carefully select antibodies based on the intended experimental model and validate reactivity in their specific system before conducting extensive studies. For cross-species comparisons, using antibodies known to recognize conserved epitopes is preferred to minimize variability unrelated to biological differences .

What are the optimized fixation and permeabilization conditions for detecting phosphorylated IRF7 using FITC-conjugated antibodies?

Detecting phosphorylated IRF7 requires specialized fixation and permeabilization protocols to preserve phosphoepitopes while enabling antibody access to nuclear antigens:

Optimized Fixation Protocol:

• First fix cells with 4% paraformaldehyde (PFA) for 15 minutes at room temperature to preserve cellular architecture

• For phosphoepitope preservation, include phosphatase inhibitors (10mM NaF, 1mM Na₃VO₄) in all buffers

• Use a gentle crosslinking fixative like methanol (-20°C, 10 minutes) as a secondary fixation step for improved nuclear antigen access

Permeabilization Considerations:
Different permeabilization methods yield varying results when detecting phosphorylated IRF7:

Critical Technical Considerations:

• The phosphorylation of Ser477 and Ser479 is essential for IRF7 activation and nuclear translocation

• Phosphatase inhibitors must be present throughout the entire protocol

• Use freshly prepared fixatives and work quickly to preserve labile phosphorylations

• Consider dual staining with total IRF7 and phospho-specific antibodies to determine the proportion of activated protein

Researchers should be aware that some fixation methods may mask the FITC signal, so optimization for each experimental system is necessary. A comparison of fixation methods using positive controls (e.g., cells treated with TLR7/9 agonists known to induce IRF7 phosphorylation) is strongly recommended .

What experimental controls are essential when studying IRF7 phosphorylation and ubiquitination status using FITC-conjugated antibodies?

Studying IRF7 post-translational modifications requires rigorous controls to ensure reliable and interpretable results:

Essential Controls for IRF7 Modification Studies:

• Positive Controls:

  • TLR7/9 stimulated pDCs (for phosphorylation)

  • Cells overexpressing IRF7 with constitutively active upstream kinases

  • Cells treated with proteasome inhibitors (MG132) to accumulate ubiquitinated IRF7 species

• Negative Controls:

  • IRF7 knockout or knockdown cells

  • Cells expressing phosphorylation-deficient IRF7 mutants (S477A/S479A)

  • Inhibitors of upstream signaling pathways (TBK1/IKKε inhibitors)

• Technical Controls:

  • Isotype-matched FITC-conjugated non-specific antibody

  • Secondary antibody-only controls

  • Phosphatase-treated samples (for phospho-specific detection)

  • DUB (deubiquitinating enzyme)-treated samples (for ubiquitination studies)

Control Experiments for Validating IRF7 Modifications:

Methodological Considerations:
TRIM21 (Ro52) cooperates with FADD to enhance ubiquitin ligase activity, promoting IRF7 ubiquitination and degradation. This creates a negative feedback loop in the IFN-α pathway . Similarly, RAUL directly catalyzes lysine 48-linked polyubiquitination of IRF7, promoting ubiquitin-proteasome dependent proteolysis .

For phosphorylation studies, researchers should note that the serine-rich domain in the Signal Response Domain (SRD) is critical, with phosphorylation of Ser477 and Ser479 being vital for IRF7 function. Substitution of these residues completely abrogates cytoplasmic to nuclear translocation .

What are the advantages and limitations of polyclonal versus monoclonal FITC-conjugated IRF7 antibodies for different applications?

Selecting between polyclonal and monoclonal FITC-conjugated IRF7 antibodies significantly impacts experimental outcomes:

Application-Specific Recommendations:

• Immunofluorescence microscopy: Monoclonal antibodies typically provide cleaner staining with less background, ideal for co-localization studies with other transcription factors

• Flow cytometry: Monoclonal antibodies offer more consistent quantification across experiments

• Western blotting: Polyclonal antibodies often provide stronger signals and can detect denatured epitopes more effectively

• ChIP applications: Monoclonal antibodies targeting the DNA-binding domain may be more effective

• Detecting modified IRF7: Polyclonal antibodies may recognize IRF7 regardless of post-translational modifications

The search results indicate availability of both monoclonal (mouse-derived, F-1 clone) and polyclonal (rabbit-derived) FITC-conjugated IRF7 antibodies , providing researchers options based on their specific experimental requirements.

What are the key considerations for researchers to remember when working with IRF7 antibodies in studying innate immune responses and autoimmune mechanisms?

Working with IRF7 antibodies requires integrating knowledge of protein biology, antibody characteristics, and disease mechanisms. Researchers should consider several critical aspects:

IRF7 functions as a master regulator of type I interferon responses, with critical implications for both antiviral immunity and autoimmune pathology . The protein's complex domain structure, including the DNA-binding domain, virus-activated domain, and signal response domain containing critical phosphorylation sites (S477/S479), influences its function and detectability by antibodies . IRF7 undergoes multiple regulatory processes including phosphorylation, ubiquitination, and nuclear translocation that must be considered in experimental design .

When selecting antibodies, researchers should match the antibody characteristics to their specific research questions. Monoclonal antibodies offer consistent specificity while polyclonal antibodies may provide stronger signals across multiple epitopes . The FITC conjugation enables direct visualization but requires protection from photobleaching and consideration of spectral overlap in multicolor experiments.

For disease-relevant research, the IRF7-IFN-I axis has been implicated in multiple autoimmune conditions including systemic lupus erythematosus, autoimmune pancreatitis, autoimmune thyroid disease, and type 1 diabetes . Genetic variants in IRF7 (SNPs rs1131665, rs1061502) have been associated with increased susceptibility to autoimmune conditions, particularly Graves' disease .

Methodologically, researchers should carefully validate antibodies in their specific experimental systems, optimize fixation and permeabilization for nuclear antigens, include appropriate positive and negative controls, and quantify results using standardized approaches that allow for reproducibility and comparison across studies.