ZFYVE1 Antibody, Biotin conjugated

What is ZFYVE1 and what are its known functions in cellular pathways?

ZFYVE1 (zinc-finger FYVE domain-containing protein 1), also known by alternative names including DFCP1, SR3, TAFF1, ZNFN2A1, and PPP1R172, is a 87kDa protein that functions primarily as a negative regulator of specific innate immune responses. Research has demonstrated that ZFYVE1 acts as a specific negative regulator of melanoma differentiation-associated gene 5 (MDA5)-mediated innate immune responses, while showing no inhibitory effect on retinoic acid-inducible gene-I (RIG-I)-mediated responses . This protein contains a guanylate-binding protein (GBP) domain and a FYVE zinc finger domain, with the N-terminal GBP domain being responsible for interactions with MDA5 . ZFYVE1 functions by binding to viral RNA and decreasing the ligand binding and oligomerization of MDA5, thereby inhibiting downstream signaling cascades that would otherwise trigger antiviral responses .

What are the key specifications of the ZFYVE1 antibody (biotin conjugated)?

The ZFYVE1 rabbit polyclonal antibody conjugated with biotin is designed for research applications with the following specifications:

• Host/Species: Rabbit

• Clonality: Polyclonal

• Conjugation: Biotin

• Tested applications: Western Blot (WB)

• Predicted reactivity: Bovine, Canine, Equine, Guinea pig, Human, Mouse, Rabbit, Rat, Zebrafish

• Immunogen: Synthetic peptide directed towards the C-terminal region of human ZFYVE1

• Form: Liquid, purified antibody supplied in 1x PBS buffer

• Molecular Weight of target protein: 87kDa

• UniProt ID: Q9HBF4

• NCBI reference: NP_067083

The antibody is directed against a synthetic peptide with the following sequence region: VCDNCYEARNVQLAVTEAQVDDEGGTLIARKVGEAVQNTLGAVVTAIDIP .

What is the optimal storage protocol for ZFYVE1 antibody to maintain its activity?

For optimal preservation of ZFYVE1 antibody activity, all biotin-conjugated antibodies should be stored in light-protected vials or covered with a light-protecting material such as aluminum foil to prevent photobleaching. These conjugated antibodies remain stable for at least 12 months when stored at 4°C . For extended storage periods up to 24 months, it is recommended to dilute the antibody with up to 50% glycerol and store at -2°C to -8°C .

It is important to note that repeated freezing and thawing cycles should be avoided as they can compromise both enzyme activity and antibody binding capabilities. The antibody is supplied in 1x PBS buffer and has an expected shelf life of 12 months from the date of receipt when properly stored .

How does ZFYVE1 function in viral immunity regulation and what experimental approaches can validate its role?

ZFYVE1 functions as a specific negative regulator of MDA5-mediated innate immune responses while having no effect on RIG-I-mediated pathways. This specificity makes it an intriguing target for investigating differential regulation of virus-sensing pathways .

Experimental approaches to validate ZFYVE1's role include:

• Gene knockout studies: ZFYVE1-deficient mice generated by CRISPR-Cas9 technology have shown enhanced protection against encephalomyocarditis virus (EMCV) which is primarily sensed by MDA5, but not against vesicular stomatitis virus (VSV) which is recognized by RIG-I .

• Protein interaction assays: Co-immunoprecipitation experiments have demonstrated that ZFYVE1 specifically interacts with MDA5 but not with RIG-I or LGP2. Domain mapping experiments revealed that the N-terminal GBP domain of ZFYVE1 is responsible for this interaction, and on MDA5, both the N-terminal tandem CARDs and the middle helicase domain can independently interact with ZFYVE1 .

• Viral RNA binding assays: "Footprint" experiments demonstrated that both ZFYVE1 and MDA5 bind to overlapping regions of EMCV RNA, suggesting competition for binding sites. ZFYVE1-deficiency increases MDA5 binding to EMCV RNA, confirming the competitive inhibition mechanism .

• Reporter assays: Overexpression of ZFYVE1 inhibits activation of the IFN-β promoter mediated by MDA5 but not by RIG-I, VISA, or IRF3, confirming its specific regulatory role in the MDA5 pathway .

What experimental controls should be implemented when using ZFYVE1 antibody in immunological research?

When conducting research with ZFYVE1 antibody, implementing appropriate controls is essential for ensuring valid and reproducible results:

• Positive control: Include cell lysates or tissues known to express ZFYVE1, such as samples from wild-type mice, particularly from tissues where innate immune responses are active.

• Negative control: Utilize ZFYVE1-deficient samples (Zfyve1-/- mice) or cell lines where ZFYVE1 has been knocked down through siRNA or CRISPR-Cas9 technology .

• Isotype control: Include a non-specific antibody of the same isotype (rabbit polyclonal with biotin conjugation) to account for non-specific binding.

• Loading control: When performing Western blot analysis, include antibodies against housekeeping proteins (β-actin, GAPDH) to normalize protein loading.

• Cross-reactivity validation: If working with non-human species, validate the antibody's cross-reactivity by comparing signal patterns between human samples and the species of interest .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide (VCDNCYEARNVQLAVTEAQVDDEGGTLIARKVGEAVQNTLGAVVTAIDIP) to confirm signal specificity .

How can ZFYVE1 antibody be utilized to investigate protein-protein interactions in innate immunity pathways?

ZFYVE1 antibody can be strategically employed to investigate protein-protein interactions in innate immunity pathways through several methodological approaches:

• Co-immunoprecipitation (Co-IP): This technique can be used to pull down ZFYVE1 and identify its binding partners. Research has shown that ZFYVE1 constitutively associates with MDA5 in uninfected cells, with this association changing upon viral infection . The biotin conjugation facilitates pull-down experiments using streptavidin-based systems.

• Proximity ligation assay (PLA): The biotinylated ZFYVE1 antibody can be paired with antibodies against potential interaction partners (e.g., MDA5) to visualize protein interactions in situ with single-molecule resolution.

• Chromatin immunoprecipitation (ChIP): For investigating potential DNA-binding activities or transcriptional regulatory functions.

• RNA immunoprecipitation (RIP): To investigate ZFYVE1's interaction with viral RNA. Studies have shown that ZFYVE1 can bind to viral RNA, competing with MDA5 for binding sites .

• Domain mapping studies: Using the antibody in conjunction with truncated protein constructs to identify specific interaction domains, as has been done to show that the N-terminal GBP domain of ZFYVE1 interacts with MDA5 .

• Protein localization studies: Using confocal microscopy to investigate co-localization of ZFYVE1 with MDA5 or other potential partners during viral infection. Studies have confirmed that ZFYVE1 co-localizes with MDA5 but not with RIG-I .

What are the optimal protocols for using biotin-conjugated ZFYVE1 antibody in Western blot applications?

For optimal Western blot results with biotin-conjugated ZFYVE1 antibody, researchers should follow these methodological guidelines:

• Sample preparation:

  • Prepare cell or tissue lysates in RIPA buffer supplemented with protease inhibitors

  • Heat samples at 95°C for 5 minutes in reducing sample buffer

  • Load 20-50 μg of total protein per lane

• Gel electrophoresis and transfer:

  • Use 8% SDS-PAGE gels (optimal for 87 kDa proteins)

  • Transfer to PVDF membrane at 100V for 90 minutes in cold transfer buffer

  • Confirm transfer efficiency with reversible protein stain

• Blocking and antibody incubation:

  • Block membrane with 5% non-fat dry milk in TBST for 1 hour at room temperature

  • Dilute primary ZFYVE1 antibody (1:500 to 1:1000) in blocking buffer

  • Incubate overnight at 4°C with gentle agitation

  • Wash 3 times with TBST, 5 minutes each

• Detection:

  • Since the antibody is biotin-conjugated, use streptavidin-HRP (1:2000 to 1:5000) in blocking buffer for 1 hour at room temperature

  • Wash 3 times with TBST, 5 minutes each

  • Develop using ECL substrate and appropriate imaging system

  • Expected band size: 87 kDa

• Optimization tips:

  • If background is high, increase blocking time or switch to 5% BSA blocking buffer

  • If signal is weak, reduce washing stringency or increase antibody concentration

  • Consider using epitope retrieval methods if the signal is difficult to detect

How can experimental design be optimized to study ZFYVE1's competitive binding with MDA5 for viral RNA?

To effectively study the competitive binding between ZFYVE1 and MDA5 for viral RNA, researchers can implement the following experimental design strategies:

• RNA pull-down assays:

  • Immobilize synthetic biotinylated viral RNA fragments or poly(I:C)-HMW on streptavidin beads

  • Incubate with cell lysates or purified proteins (MDA5, ZFYVE1, or both)

  • Analyze bound proteins by Western blot using specific antibodies

  • Varying the ratio of ZFYVE1 to MDA5 can demonstrate competitive binding

• Viral RNA "footprint" experiments:

  • Infect cells with EMCV or other viruses

  • Immunoprecipitate ZFYVE1 or MDA5 using specific antibodies

  • Extract and quantify bound viral RNA by qPCR with primers targeting different regions of the viral genome

  • Compare binding patterns between wild-type and ZFYVE1-deficient cells

• Domain mutation analysis:

  • Generate ZFYVE1 mutants lacking specific domains (e.g., ΔZF1, ΔZF2, ΔGBP)

  • Test their ability to inhibit MDA5 binding to poly(I:C)-HMW

  • Current research shows the second ZF domain of ZFYVE1 is crucial for inhibiting MDA5's RNA binding

• Fluorescence-based RNA binding assays:

  • Label viral RNA or poly(I:C) with fluorescent dyes

  • Measure binding kinetics with purified proteins using fluorescence anisotropy

  • Conduct competition assays by adding increasing concentrations of ZFYVE1 to MDA5-RNA complexes

• In vivo validation:

  • Compare viral RNA binding to MDA5 in tissues from wild-type versus Zfyve1-/- mice following viral infection

  • Analyze downstream signaling activation in both genotypes

What are the potential sources of data variability when studying ZFYVE1 function in innate immunity, and how can they be addressed?

When investigating ZFYVE1's function in innate immunity, several sources of data variability may arise. Understanding and addressing these factors is crucial for generating reliable and reproducible results:

• Variable viral infection efficiency:

  • Standardize viral stocks by plaque assay or TCID50

  • Use MOI (multiplicity of infection) calculations to ensure consistent infection rates

  • Include positive controls (e.g., known viral response genes) to confirm infection

  • Solution: Normalize data to viral load measured by qPCR

• Differences in ZFYVE1 expression levels:

  • Basal expression of ZFYVE1 may vary between cell types and experimental conditions

  • Solution: Quantify ZFYVE1 levels by Western blot or qPCR and normalize results accordingly

• Timing of measurements:

  • The association between ZFYVE1 and MDA5 changes dynamically during viral infection

  • Solution: Conduct time-course experiments and specify exact time points for measurements

• Technical variation in antibody-based assays:

  • Batch-to-batch variation in antibody performance

  • Solution: Use consistent antibody lots, include standard controls in each experiment

• Genetic background effects in knockout models:

  • ZFYVE1-deficient mice may have compensatory mechanisms affecting results

  • Solution: Use littermate controls and validate findings in multiple independent knockout lines

• Cell type-specific effects:

  • ZFYVE1's regulatory function may vary between cell types

  • Solution: Compare results across multiple relevant cell types (e.g., MLFs, BMDCs)

• Viral strain specificity:

  • ZFYVE1's effects are virus-dependent (e.g., affects EMCV but not VSV responses)

  • Solution: Test multiple virus types recognized by different pattern recognition receptors

How do experimental findings on ZFYVE1 translate to potential therapeutic approaches for viral infections?

The experimental findings on ZFYVE1's role in regulating MDA5-mediated innate immune responses suggest several potential therapeutic strategies for viral infections:

• Targeted ZFYVE1 inhibition:

  • Temporary inhibition of ZFYVE1 function could enhance MDA5-mediated antiviral responses against specific viruses sensed primarily by MDA5 (like EMCV)

  • This approach is supported by evidence that ZFYVE1-deficient mice showed enhanced protection against EMCV-induced lethality

  • Small molecule inhibitors or peptide-based approaches targeting the interaction between ZFYVE1 and MDA5 could be developed

• Pathway-specific immune modulation:

  • Since ZFYVE1 specifically regulates MDA5 but not RIG-I pathways, targeting ZFYVE1 provides a way to selectively enhance specific antiviral responses

  • This selective approach could minimize broad immunopathology associated with non-specific immune activation

• Biomarkers for viral susceptibility:

  • ZFYVE1 expression levels or polymorphisms could potentially serve as biomarkers for susceptibility to certain viral infections

  • Personalized therapeutic approaches could be developed based on ZFYVE1 status

• Adjuvant development:

  • Transient ZFYVE1 inhibition could potentially serve as an adjuvant strategy for vaccines against viruses recognized by MDA5

  • Enhanced MDA5 signaling could amplify immune responses to vaccination

• Cell-type specific targeting:

  • Since ZFYVE1 functions in multiple cell types including lung fibroblasts and dendritic cells, cell-type specific delivery systems could be developed to target ZFYVE1 in relevant tissues during infection

What comparative analysis techniques are useful when evaluating ZFYVE1 antibody specificity across different experimental systems?

When evaluating ZFYVE1 antibody specificity across different experimental systems, several comparative analysis techniques can be employed:

This comparative approach ensures robust validation of antibody specificity across different experimental conditions, which is crucial for generating reliable data, especially when studying a regulatory protein like ZFYVE1 that has multiple domains and interaction partners .

How can researchers reconcile contradictory data regarding ZFYVE1 function in different experimental models?

Reconciling contradictory data regarding ZFYVE1 function across different experimental models requires systematic analysis and careful consideration of multiple factors:

What emerging technologies could enhance the study of ZFYVE1's role in innate immunity regulation?

Several emerging technologies hold promise for advancing our understanding of ZFYVE1's role in innate immunity regulation:

• CRISPR-based screening approaches:

  • Genome-wide CRISPR screens can identify novel interaction partners or regulatory factors affecting ZFYVE1 function.

  • CRISPRi/CRISPRa systems allow for temporal control of ZFYVE1 expression to study dynamic responses.

  • Base editing techniques can introduce specific mutations to study structure-function relationships.

• Single-cell technologies:

  • Single-cell RNA-seq can reveal cell-type specific responses to ZFYVE1 modulation during viral infection.

  • Single-cell proteomics may uncover heterogeneity in ZFYVE1 expression and function across immune cell populations.

  • Spatial transcriptomics can map ZFYVE1 activity within tissues during infection.

• Advanced imaging techniques:

  • Super-resolution microscopy can visualize ZFYVE1-MDA5 interactions at nanometer resolution.

  • Live-cell imaging with fluorescently tagged proteins can track the dynamics of ZFYVE1-MDA5 interactions during viral infection.

  • Correlative light and electron microscopy (CLEM) can link protein interactions to ultrastructural changes.

• Structural biology approaches:

  • Cryo-EM studies of ZFYVE1-MDA5 complexes with viral RNA can provide detailed structural insights.

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) can map conformational changes upon binding.

  • AlphaFold or similar AI-based structure prediction tools can model interaction interfaces.

• Systems biology integration:

  • Multi-omics approaches combining transcriptomics, proteomics, and metabolomics data can provide a comprehensive view of ZFYVE1's impact.

  • Mathematical modeling of innate immune signaling networks incorporating ZFYVE1's regulatory effects.

  • Network analysis to position ZFYVE1 within the broader immune signaling landscape.

• Organoid and animal models:

  • Human immune organoids can bridge the gap between cell culture and in vivo studies.

  • Humanized mouse models expressing human ZFYVE1 can better translate findings to human biology.

  • Tissue-specific conditional knockout models can dissect ZFYVE1's role in different anatomical compartments.

How can researchers design experiments to further elucidate the domain-specific functions of ZFYVE1 in immune regulation?

To further elucidate the domain-specific functions of ZFYVE1 in immune regulation, researchers can design comprehensive experimental approaches:

• Domain deletion and mutation studies:

  • Generate a comprehensive panel of domain deletion mutants (ΔGBP, ΔZF1, ΔZF2) and point mutations within each domain.

  • Assess each mutant's ability to:

    • Bind MDA5 (co-immunoprecipitation)

    • Compete for viral RNA binding (RNA pull-down)

    • Inhibit IFN-β promoter activation (reporter assays)

    • Localize properly within cells (confocal microscopy)

• Domain swap experiments:

  • Create chimeric proteins by swapping domains between ZFYVE1 and related proteins.

  • Test if the GBP domain from ZFYVE1 confers MDA5-binding ability to other proteins.

  • Determine if the zinc finger domains from other proteins can substitute for ZFYVE1's RNA-binding function.

• Structure-function analysis:

  • Employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify regions that undergo conformational changes upon binding to MDA5 or viral RNA.

  • Use crosslinking mass spectrometry to map specific interaction residues between ZFYVE1 domains and their binding partners.

• Domain-specific knockin mouse models:

  • Generate knockin mice expressing ZFYVE1 with specific domain mutations.

  • Challenge these mice with viruses recognized by MDA5 versus RIG-I to assess domain-specific contributions to viral defense in vivo .

• Temporal regulation studies:

  • Develop inducible expression systems for domain-specific mutants.

  • Analyze how different domains contribute to the dynamic association between ZFYVE1 and MDA5 during the course of viral infection .

• Interactome mapping:

  • Perform BioID or APEX proximity labeling using domain-specific mutants as baits.

  • Identify domain-specific interaction partners that may contribute to ZFYVE1's regulatory functions.

• Competitive inhibition studies:

  • Develop peptides or small molecules targeting specific ZFYVE1 domains.

  • Test their effects on MDA5-mediated signaling in cellular and animal models of viral infection.