ZFYVE1 Antibody, HRP conjugated

What is ZFYVE1 and what cellular functions does it perform?

ZFYVE1 (also known as DFCP1, SR3, TAFF1) is a zinc-finger protein containing two FYVE domains in tandem that primarily localizes to the Golgi apparatus and Golgi stacks . The protein plays critical roles in multiple cellular processes:

• Autophagy: ZFYVE1 is recruited to PtdIns3P-containing domains of the endoplasmic reticulum during autophagosome biogenesis, serving as a marker for omegasome formation .

• Innate immunity: ZFYVE1 functions as a positive regulator of TLR3-mediated signaling by promoting the binding of TLR3 to its ligand, such as viral dsRNA and synthetic poly(I:C) .

• Membrane trafficking: The FYVE domains of ZFYVE1 mediate recruitment to phosphatidylinositol 3-phosphate-containing membranes, contributing to cellular membrane dynamics .

The protein is expressed in multiple tissues and can be detected in human and mouse samples using appropriate antibodies .

What are the key structural features and domains of ZFYVE1?

ZFYVE1 contains several important structural domains that facilitate its cellular functions:

• Tandem FYVE domains: ZFYVE1 contains two zinc-binding FYVE domains in sequence, which are responsible for binding to PtdIns3P on membrane surfaces .

• Zinc finger motifs: These contribute to the protein's ability to interact with membranes and potentially other proteins.

• Alternative splice variants: The ZFYVE1 gene can produce multiple transcript variants through alternative splicing, potentially resulting in proteins with different functional properties .

The FYVE domains are particularly significant as they enable ZFYVE1 to be recruited to PtdIns3P-enriched membranes during critical cellular processes like autophagy initiation .

Why choose HRP-conjugated ZFYVE1 antibodies for research applications?

HRP (horseradish peroxidase)-conjugated antibodies offer several advantages for ZFYVE1 detection:

• Direct detection: HRP-conjugated primary antibodies eliminate the need for secondary antibodies, simplifying protocols and reducing background noise.

• Enhanced sensitivity: The enzymatic amplification provided by HRP allows for detection of low-abundance ZFYVE1, particularly in tissues where expression may be limited.

• Quantitative analysis: HRP-conjugated antibodies provide consistent signal intensity that correlates with protein abundance, facilitating quantitative assessment.

• Multiplex compatibility: When studying ZFYVE1 alongside other proteins, HRP-conjugated antibodies can be paired with fluorescent detection methods for simultaneous analysis.

While unconjugated ZFYVE1 antibodies (like the rabbit polyclonal from Elabscience) can be used with HRP-conjugated secondary antibodies , directly conjugated antibodies streamline workflows for techniques like Western blotting and immunohistochemistry.

What are the common synonyms and designations for ZFYVE1?

When searching literature or antibody databases, researchers should be aware of these alternative designations for ZFYVE1:

These alternative names may appear in different research contexts, with DFCP1 being particularly common in autophagy research literature .

What are the optimal dilution ratios for ZFYVE1 antibodies in different applications?

The dilution ratios for ZFYVE1 antibodies vary depending on the application and the specific antibody formulation:

When using HRP-conjugated ZFYVE1 antibodies, dilutions may differ from unconjugated versions. Always perform a dilution series to determine optimal concentration for your specific experimental system .

How should samples be prepared for optimal ZFYVE1 detection?

Sample preparation is critical for successful ZFYVE1 detection:

For tissue sections (IHC):

• Fixation: 10% neutral buffered formalin is commonly used, but paraformaldehyde may better preserve ZFYVE1 epitopes.

• Antigen retrieval: Heat-induced epitope retrieval using citrate buffer (pH 6.0) is recommended.

• Blocking: Use 5-10% normal serum from the same species as the secondary antibody to reduce non-specific binding.

For cell cultures:

• Fixation: 4% paraformaldehyde (10-15 minutes) generally preserves ZFYVE1 localization.

• Permeabilization: 0.1-0.5% Triton X-100 for adequate antibody access to intracellular ZFYVE1.

• Storage: Fixed cells can be stored at 4°C in PBS with 0.05% sodium azide, but fresh samples are preferred .

For Western blotting:

• Lysis buffer selection: RIPA buffer with protease inhibitors is appropriate for ZFYVE1 extraction.

• Sample handling: Avoid repeated freeze-thaw cycles of protein lysates to prevent degradation.

• Loading controls: Include appropriate controls (β-actin, GAPDH) for normalization .

How can I validate the specificity of a ZFYVE1 antibody?

Validation of ZFYVE1 antibody specificity is crucial for reliable experimental results:

• Positive controls: Use tissues or cell lines known to express ZFYVE1, such as human tonsil samples for IHC applications .

• Negative controls:

  • Omit primary antibody but include all other reagents

  • Test in ZFYVE1-deficient cells or tissues (knockout models)

• Blocking peptide: Pre-incubate the antibody with the immunizing peptide (if available) to confirm signal elimination.

• Multiple antibodies: Compare staining patterns using antibodies targeting different epitopes of ZFYVE1.

• Molecular weight verification: In Western blots, confirm band appears at the expected molecular weight.

• siRNA knockdown: Reduced signal following ZFYVE1 siRNA treatment confirms specificity .

• Recombinant expression: Compare endogenous pattern with overexpressed tagged ZFYVE1 .

What are the recommended storage conditions for preserving ZFYVE1 antibody activity?

Proper storage is essential for maintaining ZFYVE1 antibody performance:

• Temperature: Store at -20°C for long-term storage (valid for 12 months under proper conditions) .

• Buffer conditions: Typically stored in phosphate buffered solution (pH 7.4) containing a stabilizer and 50% glycerol .

• Aliquoting: Divide into small working aliquots to avoid repeated freeze-thaw cycles.

• Thawing procedure: Thaw completely at room temperature, then keep on ice during experiments.

• Shipping considerations: The product is typically shipped with ice packs and should be stored immediately at -20°C upon receipt .

• Working solution: Once diluted, use within 24 hours and store at 4°C.

How does ZFYVE1 contribute to autophagosome formation and how can this be studied?

ZFYVE1/DFCP1 plays a critical role in autophagosome biogenesis:

• Mechanism: ZFYVE1 is recruited to PtdIns3P-rich domains on the endoplasmic reticulum (ER) at sites where autophagosome formation is initiated, serving as a marker for omegasomes .

• Experimental approaches:

  • Colocalization studies with autophagy markers: ZFYVE1 can be detected at sites also stained with LC3 and WIPI2, indicating its role in early autophagosome formation .

  • Time-course experiments: ZFYVE1 recruitment precedes LC3 at autophagosome formation sites .

  • PtdIns3P detection: GST-FYVE probes can be used alongside ZFYVE1 staining to confirm PtdIns3P-rich membrane domains .

• Methodological considerations:

  • Fixed versus live imaging: While GFP-tagged ZFYVE1 provides dynamic information, it may alter membrane stability; indirect GST-FYVE staining in fixed cells offers an alternative approach .

  • Inhibitor studies: PIK3C3 inhibitors like 3-methyladenine (3MA) and wortmannin can be used to confirm ZFYVE1 recruitment dependency on PtdIns3P production .

What is the role of ZFYVE1 in TLR3-mediated immune signaling?

ZFYVE1 functions as a positive regulator of TLR3-mediated innate immune responses:

• Functional impact:

  • Overexpression of ZFYVE1 promotes transcription of antiviral genes after poly(I:C) stimulation .

  • ZFYVE1 deficiency inhibits poly(I:C)-induced activation of IFN-β promoter, ISRE, and NF-κB .

  • ZFYVE1 knockout mice show reduced susceptibility to inflammatory death induced by poly(I:C) .

• Molecular mechanisms:

  • ZFYVE1 associates with TLR3 through its FYVE domain interacting with the TLR3 ectodomain .

  • ZFYVE1 binds to poly(I:C) and increases TLR3's binding affinity to this ligand .

  • The association between ZFYVE1 and TLR3 increases upon poly(I:C) stimulation .

• Experimental approaches:

  • Co-immunoprecipitation: To detect interaction between ZFYVE1 and TLR3 .

  • Phosphorylation assays: To monitor downstream signaling events (TBK1, IRF3, p65 phosphorylation) .

  • Gene expression analysis: qPCR for IFNB1, ISG56, and CXCL1 to assess signaling pathway activation .

How can ZFYVE1 knockout or knockdown models be developed and validated?

Creating and validating ZFYVE1-deficient models:

• CRISPR/Cas9 method:

  • Successfully used to generate ZFYVE1-deficient HT1080 and 293-TLR4 cells .

  • Validation involves Western blot confirmation of protein absence and functional assays showing impaired poly(I:C)-induced signaling .

• RNAi approaches:

  • Multiple ZFYVE1-RNAi constructs can be tested for knockdown efficiency .

  • Validation through Western blot and functional readouts like reporter assays (IFN-β promoter, ISRE, NF-κB) .

• Mouse models:

  • Zfyve1-/- mice show reduced susceptibility to poly(I:C)-induced inflammatory death .

  • Primary cells (MLFs, BMDCs) from these mice display impaired poly(I:C)-induced transcription of antiviral genes .

• Validation controls:

  • Include pathway-specific controls (LPS stimulation remains unaffected in ZFYVE1-deficient models) .

  • Rescue experiments with ZFYVE1 re-expression to confirm phenotype specificity.

What techniques can be used to study ZFYVE1 protein-protein interactions?

Several approaches can investigate ZFYVE1's interactions with other proteins:

• Co-immunoprecipitation (Co-IP):

  • Endogenous Co-IP demonstrates ZFYVE1 association with TLR3 .

  • Time-course experiments reveal how interactions change with stimulation (e.g., poly(I:C)) .

• Domain mapping:

  • The FYVE domain of ZFYVE1 and the ectodomain of TLR3 are responsible for their interaction .

  • Truncation or mutation constructs can pinpoint exact interaction regions.

• Confocal microscopy:

  • Shows colocalization of ZFYVE1 with binding partners like TLR3 .

  • Can be combined with stimulation experiments to track dynamic interactions .

• Proximity ligation assay (PLA):

  • Provides high-sensitivity detection of protein-protein interactions in situ.

  • Useful for detecting transient or weak interactions that might be lost in Co-IP.

• High-throughput screening approaches:

  • Mammalian two-hybrid systems like MAPPIT and KISS can identify novel ZFYVE1 interaction partners .

What are the expected staining patterns for ZFYVE1 in different subcellular compartments?

ZFYVE1 exhibits distinct localization patterns that vary with cellular context:

• Basal conditions:

  • Predominantly localized to Golgi apparatus and Golgi stacks .

  • Punctate cytoplasmic staining pattern in immunofluorescence.

• During autophagy:

  • Recruitment to PtdIns3P-positive domains on the ER .

  • Colocalization with or proximity to LC3-positive structures .

  • Discrete puncta that may be adjacent to WIPI2-positive structures .

• During TLR3 activation:

  • Partial colocalization with TLR3 that increases following poly(I:C) stimulation .

  • Dynamic association with signaling complexes containing components like Mex3B .

When interpreting staining patterns, consider that overexpressed tagged ZFYVE1 may show different distribution compared to endogenous protein detected by antibodies .

How can non-specific binding be distinguished from true ZFYVE1 signals?

Distinguishing specific from non-specific signals:

• Pattern recognition:

  • True ZFYVE1 signal shows Golgi enrichment and/or distinct punctate structures .

  • Non-specific binding often appears as diffuse cytoplasmic or nuclear staining.

• Control experiments:

  • ZFYVE1 knockout/knockdown samples should show significantly reduced signal .

  • Blocking peptide competition should eliminate specific but not non-specific signals.

  • Secondary-only controls identify background from the detection system.

• Signal characteristics:

  • Western blot: Specific band at the expected molecular weight (~80-90 kDa).

  • IHC/IF: Signal in expected subcellular locations and cell types known to express ZFYVE1.

• Titration effects:

  • True signals typically show concentration-dependent intensity when antibody is diluted.

  • Non-specific binding may persist even at high dilutions.

What are common troubleshooting approaches for weak or absent ZFYVE1 signal?

When ZFYVE1 detection is suboptimal:

• Sample preparation issues:

  • Ensure proper fixation that preserves ZFYVE1 epitopes (4% paraformaldehyde recommended).

  • Try different antigen retrieval methods for IHC (citrate buffer pH 6.0 vs. EDTA pH 9.0).

  • Check protein extraction efficiency with different lysis buffers.

• Antibody factors:

  • Verify antibody viability (avoid repeated freeze-thaw cycles) .

  • Optimize dilution (try a range from 1:30 to 1:150 for IHC) .

  • Consider different antibody clones that target different epitopes.

• Detection system:

  • For HRP-conjugated antibodies, ensure substrate is fresh and properly prepared.

  • Extend development/exposure time to detect low abundance signals.

  • For fluorescent detection, use higher sensitivity settings and minimize photobleaching.

• Biological considerations:

  • Confirm ZFYVE1 expression in your experimental system.

  • Consider treatments that upregulate ZFYVE1 (e.g., autophagy induction).

  • Some tissues naturally express lower levels and may require more sensitive detection methods.

How can ZFYVE1 antibodies be used to monitor autophagy and TLR3 signaling dynamics?

ZFYVE1 antibodies can track key cellular processes:

• Autophagy monitoring:

  • Time-course experiments to track ZFYVE1 recruitment to PtdIns3P-positive structures .

  • Co-staining with autophagy markers (LC3, WIPI2) to identify sequential recruitment .

  • Comparison of starvation-induced versus drug-induced autophagy effects on ZFYVE1 localization.

• TLR3 signaling analysis:

  • Detection of ZFYVE1-TLR3 complexes at different time points after poly(I:C) stimulation .

  • Correlation of ZFYVE1-TLR3 interaction with downstream signaling events (IRF3/p65 phosphorylation) .

  • Comparison between wild-type and ZFYVE1-deficient cells to quantify signaling differences .

• Quantitative approaches:

  • Count ZFYVE1-positive puncta per cell to measure autophagy induction.

  • Measure colocalization coefficients between ZFYVE1 and interaction partners.

  • Analyze western blot band intensity to quantify expression changes during cellular responses.

• High-content imaging:

  • Automated image analysis of ZFYVE1 puncta formation across multiple conditions.

  • Single-cell analysis to account for heterogeneous responses in cell populations.