ZFYVE9 Antibody, Biotin conjugated

What is ZFYVE9 and what are its primary functions in cellular signaling?

ZFYVE9 (Zinc Finger FYVE-type Containing 9) is a double zinc finger motif-containing protein that plays a crucial role in the transforming growth factor-beta (TGF-β) signaling pathway. Also known as SARA (Smad Anchor for Receptor Activation), NSP, MADHIP, SMADIP, or PPP1R173, this protein interacts directly with SMAD2 and SMAD3, recruiting SMAD2 to the TGF-β receptor . This recruitment function is essential for proper signal transduction from the cell membrane to the nucleus. The ZFYVE9 gene is located on chromosome 1p32.3 and contains 21 exons . Its involvement in TGF-β signaling makes ZFYVE9 significant in development, tissue homeostasis, and various pathological conditions including fibrosis and cancer progression, where TGF-β signaling is frequently dysregulated.

What applications are most suitable for biotin-conjugated ZFYVE9 antibodies?

Biotin-conjugated ZFYVE9 antibodies are particularly valuable for several experimental applications. The primary application is ELISA, where the biotin conjugation enables sensitive detection when paired with streptavidin-conjugated reporter molecules . Additional applications include immunofluorescence studies, where the biotin tag allows for signal amplification and flexibility in detection systems. When selecting a biotin-conjugated ZFYVE9 antibody, researchers should verify that the specific product has been validated for their intended application. For example, the ABIN7176069 antibody has been validated specifically for ELISA applications and targets the amino acid region 683-926 of human ZFYVE9 . The biotin conjugation provides advantages in signal amplification and detection flexibility compared to unconjugated antibodies.

How should I select the appropriate binding specificity for my ZFYVE9 antibody research?

When selecting a ZFYVE9 antibody, researchers must consider several factors to ensure appropriate binding specificity for their experimental needs. First, evaluate the target epitope region - different antibodies target distinct regions of ZFYVE9, such as AA 683-926, AA 1-762, AA 71-170, AA 1-260, or the C-terminal region . This choice should align with your research question, particularly if you're interested in specific domains or isoforms. Second, confirm species reactivity matches your experimental model - some ZFYVE9 antibodies react only with human samples, while others cross-react with mouse and rat proteins . Third, verify the antibody has been validated for your intended application (WB, ELISA, IF, etc.). Finally, consider clonality - polyclonal antibodies like ABIN7176068 and ABIN7176069 recognize multiple epitopes and may provide stronger signals, while monoclonal antibodies offer higher specificity . For domain-specific studies (such as the FYVE domain), select antibodies targeting those particular regions.

What controls should I include when using biotin-conjugated ZFYVE9 antibodies?

When using biotin-conjugated ZFYVE9 antibodies, implementing proper controls is essential for experimental validity. Primary controls should include a positive control (cell line or tissue known to express ZFYVE9), a negative control (preferably ZFYVE9 knockout or knockdown samples), and a blocking peptide control (pre-incubation of the antibody with the immunizing peptide). Secondary controls must include a streptavidin-only control to assess endogenous biotin, which is particularly important as endogenous biotin can cause background issues in certain tissues and cell types . For biotin-conjugated antibodies like ABIN7176069, include a titration series to determine optimal concentration for specific detection without background . Additionally, include isotype controls (matched IgG) to evaluate non-specific binding. When performing co-localization studies, include single-staining controls to assess bleed-through. Finally, technical controls should test different fixation and permeabilization methods, as these can significantly affect epitope accessibility and biotin-streptavidin interaction efficiency.

How do biotin-conjugated ZFYVE9 antibodies compare to unconjugated versions for various applications?

Biotin-conjugated ZFYVE9 antibodies offer distinct advantages and limitations compared to their unconjugated counterparts across various applications. For ELISA applications, biotin-conjugated antibodies (such as ABIN7176069) provide superior signal amplification through the streptavidin-biotin system, resulting in enhanced sensitivity . This makes them particularly valuable for detecting low-abundance ZFYVE9 protein. In contrast, unconjugated antibodies (like ABIN7176068) offer greater versatility across multiple applications including Western Blotting, ELISA, and Immunofluorescence . For immunofluorescence studies, biotin-conjugated antibodies enable multi-step detection strategies and signal amplification but may introduce higher background from endogenous biotin. Unconjugated antibodies allow more direct detection with secondary antibodies. When considering Western Blotting, unconjugated antibodies typically perform better as they can be paired with various secondary antibody systems, while biotin-conjugated versions may have limited utility unless specifically validated for this application. The choice between biotin-conjugated and unconjugated formats should be guided by the specific application, required sensitivity, and background considerations in your experimental system.

What methodological approaches optimize detection of ZFYVE9 in subcellular compartments?

Detecting ZFYVE9 in different subcellular compartments requires optimized methodological approaches due to its dynamic localization patterns. ZFYVE9 contains a FYVE domain that interacts with phosphatidylinositol 3-phosphate (PI3P) in endosomal membranes, similar to the mechanism observed with ZFYVE1 . For endosomal localization studies, mild fixation (2-4% PFA for 10-15 minutes) preserves membrane structures better than methanol fixation. Co-staining with endosomal markers (Rab5, EEA1) helps distinguish specific endosomal populations. For membrane vs. cytosolic distribution, cellular fractionation studies have shown that ZFYVE9, like related FYVE-domain proteins, can be detected in membrane, cytosolic, and even mitochondrial fractions . When using biotin-conjugated antibodies like ABIN7176069, optimize permeabilization conditions (0.1-0.3% Triton X-100) to maintain epitope accessibility while allowing antibody penetration . For stimulus-dependent relocalization studies, time-course experiments with synchronized TGF-β stimulation are essential, as FYVE domain proteins show dynamic translocation from cytosolic to membrane fractions upon stimulation . Confocal microscopy with z-stack acquisition is crucial for accurate localization assessment, particularly when evaluating potential nuclear localization.

How can I systematically troubleshoot non-specific binding with biotin-conjugated ZFYVE9 antibodies?

Systematic troubleshooting of non-specific binding with biotin-conjugated ZFYVE9 antibodies requires a methodical approach addressing multiple potential issues. First, address endogenous biotin interference by implementing a biotin blocking step using commercially available endogenous biotin blocking kits before applying the biotin-conjugated ZFYVE9 antibody . Second, optimize blocking conditions by testing different blocking agents (BSA vs. milk vs. serum) and increasing blocking time (2-3 hours at room temperature or overnight at 4°C). Third, titrate the primary antibody concentration, starting with the manufacturer's recommended dilution for ABIN7176069 and testing 2-3 dilutions above and below this range . Fourth, optimize streptavidin-conjugate concentration, as excess streptavidin can bind non-specifically to biotin-like structures. Fifth, increase wash stringency by adding additional wash steps and increasing detergent concentration in wash buffers (0.1-0.5% Tween-20 or Triton X-100). Sixth, validate specificity using peptide competition assays where pre-incubation of the antibody with immunizing peptide should eliminate specific signal. Finally, compare staining patterns with alternative ZFYVE9 antibodies targeting different epitopes to distinguish between specific and non-specific signals. Document each optimization step systematically to establish a reproducible protocol.

What protocol modifications are necessary when using ZFYVE9 antibodies for co-localization studies with TGF-β pathway components?

When conducting co-localization studies of ZFYVE9 with TGF-β pathway components, several critical protocol modifications are necessary for optimal results. First, antibody selection is crucial - when using biotin-conjugated ZFYVE9 antibodies like ABIN7176069, ensure secondary detection reagents are compatible with other primary antibodies in the multiplexing experiment . For detecting interactions with SMAD2/3, consider antibodies targeting the amino acid region 683-926 of ZFYVE9, as this contains domains involved in SMAD interactions . Second, fixation and permeabilization must be optimized - 4% paraformaldehyde followed by 0.1-0.2% Triton X-100 permeabilization typically preserves both membrane structures and protein-protein interactions. Third, implement sequential staining when using biotin-conjugated antibodies - complete the biotin-streptavidin detection first, followed by substantial washing before introducing other antibodies to prevent cross-reactivity. Fourth, for temporal dynamics studies, synchronize cells with serum starvation (6-12 hours) before TGF-β stimulation, then fix cells at multiple timepoints (5, 15, 30, 60 minutes) to capture the dynamic recruitment of SMAD proteins by ZFYVE9. Finally, employ appropriate imaging technology - confocal microscopy with spectral unmixing capabilities is essential to accurately distinguish fluorophores and assess true co-localization versus signal overlap.

How can ZFYVE9 antibodies be employed to investigate the role of FYVE domain in membrane targeting and protein function?

Investigating the FYVE domain of ZFYVE9 using specific antibodies requires sophisticated experimental approaches that leverage the domain's structural and functional properties. The FYVE domain in ZFYVE9, similar to that in related proteins like ZFYVE1, mediates binding to phosphatidylinositol 3-phosphate (PI3P) in endosomal membranes . To study this domain specifically, researchers can implement domain-focused approaches using ZFYVE9 antibodies. Begin with subcellular fractionation studies combined with Western blotting using antibodies targeting different regions of ZFYVE9 to track the distribution between cytosolic and membrane fractions before and after TGF-β stimulation . For mutation analysis, compare wild-type ZFYVE9 localization with FYVE domain mutants (similar to ZFYVE1(C654/770S) or ZFYVE1(W543A) models) that disrupt membrane binding . Implement live-cell imaging with biotin-tagged ZFYVE9 antibody fragments (Fab) combined with fluorescent streptavidin to track dynamic recruitment to endosomal membranes in real-time. For structure-function analysis, complement antibody studies with proteinase K protection assays to determine the topology of ZFYVE9 insertion into membranes, as demonstrated with related FYVE-domain proteins . Proximity ligation assays using ZFYVE9 antibodies with antibodies against PI3P or endosomal markers can visualize specific interaction sites within cells.

What methodological approaches can reveal the dynamical relationship between ZFYVE9 and SMAD proteins during TGF-β signaling?

Revealing the dynamic relationship between ZFYVE9 and SMAD proteins during TGF-β signaling requires sophisticated methodological approaches. Temporal interaction studies can be conducted using co-immunoprecipitation with ZFYVE9 antibodies (such as ABIN7176068) at defined time points after TGF-β stimulation (0, 5, 15, 30, 60 minutes), followed by Western blotting for SMAD2/3 to track association kinetics . For spatial dynamics, implement live-cell imaging using biotin-conjugated ZFYVE9 antibody fragments (Fab) combined with fluorescent streptavidin in cells expressing fluorescently-tagged SMAD proteins. This approach allows visualization of recruitment events at the membrane and subsequent dissociation. Proximity ligation assays (PLA) using ZFYVE9 antibodies together with SMAD2/3 antibodies can provide single-molecule sensitivity for detecting transient interactions in fixed cells. For functional impact assessment, combine siRNA-mediated ZFYVE9 knockdown with phospho-SMAD2/3 immunostaining to evaluate how ZFYVE9 depletion affects SMAD phosphorylation kinetics and nuclear translocation. FRET/FLIM analysis between labeled ZFYVE9 antibodies and SMAD proteins can measure direct interactions with nanometer resolution. Finally, develop phospho-specific ZFYVE9 antibodies to determine how ZFYVE9 phosphorylation states correlate with its ability to recruit and activate SMAD proteins during signaling events.

How can researcher-developed validation protocols verify the specificity of commercial ZFYVE9 antibodies for advanced applications?

Developing comprehensive validation protocols for commercial ZFYVE9 antibodies requires multi-layered approaches that extend beyond manufacturer specifications. Begin with genetic validation by testing the antibody in ZFYVE9 knockdown/knockout models generated via CRISPR/Cas9 or siRNA methods - complete elimination or significant reduction of signal confirms specificity . For antibodies like ABIN7176068 and ABIN7176069, which target the amino acid region 683-926, express this specific fragment in a heterologous system as a positive control . Implement cross-reactivity assessment by testing the antibody against related FYVE-domain proteins, particularly when using the antibody in non-human samples. For application-specific validation, perform peptide competition assays where pre-incubation with the immunizing peptide should eliminate specific signal in each application (WB, IF, ELISA). For biotin-conjugated antibodies like ABIN7176069, develop specific controls for biotin-related background by comparing signals in tissues with high versus low endogenous biotin levels . Implement orthogonal validation by comparing detection with antibodies targeting different epitopes of ZFYVE9 and correlating results with mRNA expression data. For post-translational modification sensitivity, test antibody recognition under conditions that alter ZFYVE9 phosphorylation or ubiquitination status. Finally, perform immunoprecipitation followed by mass spectrometry to confirm that the antibody specifically enriches ZFYVE9 and known interacting partners.

What protocol adaptations enable ZFYVE9 antibodies to elucidate cross-talk between TGF-β signaling and other pathways?

Elucidating cross-talk between TGF-β signaling and other pathways using ZFYVE9 antibodies requires specialized protocol adaptations. For simultaneous pathway stimulation experiments, implement dual-treatment protocols where cells are pre-treated with modulators of secondary pathways (e.g., Wnt, Notch, MAPK inhibitors/activators) before TGF-β stimulation, then use ZFYVE9 antibodies like ABIN7176068 to track changes in localization or interaction patterns . For multi-pathway protein complex analysis, develop sequential immunoprecipitation protocols where ZFYVE9 is first immunoprecipitated using specific antibodies, followed by elution and second immunoprecipitation with antibodies against components of intersecting pathways. Implement multiplexed proximity ligation assays (PLA) using combinations of ZFYVE9 antibodies with antibodies against proteins from different signaling pathways to visualize pathway nodes at endogenous levels. For pathway-dependent phosphorylation studies, combine phosphatase inhibitor treatments with ZFYVE9 immunoprecipitation followed by phospho-specific Western blotting or mass spectrometry to identify pathway-specific phosphorylation events. Develop multi-epitope detection strategies where biotin-conjugated ZFYVE9 antibodies like ABIN7176069 are used alongside antibodies against proteins from other pathways using orthogonal detection systems . Finally, adapt ChIP-seq protocols using ZFYVE9 antibodies to identify genomic loci where ZFYVE9-associated complexes might integrate signals from multiple pathways, particularly at genes known to respond to multiple signaling inputs.

What quantitative methods best analyze ZFYVE9 expression data from immunoblotting experiments?

Quantitative analysis of ZFYVE9 expression from immunoblotting experiments requires rigorous methodological approaches to ensure accuracy and reproducibility. Begin with proper experimental design including technical replicates (minimum three per condition) and appropriate controls using ZFYVE9 antibodies validated for Western blotting, such as ABIN7176068 . For densitometric analysis, use specialized software (ImageJ, Image Studio, etc.) to quantify band intensity while ensuring signals fall within the linear range of detection. Implement normalization strategies by first normalizing ZFYVE9 signal to loading controls (β-actin, GAPDH, α-tubulin), then to the experimental control condition. For detecting multiple isoforms, perform isoform-specific quantification by measuring each band separately and reporting individual values along with total protein. When comparing across multiple blots, include a common reference sample on each blot for inter-blot normalization. For statistical analysis, apply appropriate tests based on data distribution (t-test for two conditions, ANOVA for multiple conditions) after confirming normality. Calculate and report both effect size and p-values. For temporal expression studies, develop time-course normalization where each timepoint is presented relative to both baseline (time 0) and maximum expression. Finally, present quantitative data in graphical format alongside representative blot images, clearly indicating molecular weight markers and using consistent axis scaling across comparable experiments.

How should researchers standardize and interpret immunofluorescence data when using biotin-conjugated ZFYVE9 antibodies?

Standardizing and interpreting immunofluorescence data with biotin-conjugated ZFYVE9 antibodies requires rigorous methodological controls and quantitative approaches. Begin with acquisition standardization by establishing fixed exposure settings based on positive and negative controls for each experiment using biotin-conjugated antibodies like ABIN7176069 . Implement flat-field correction to compensate for uneven illumination, particularly important when quantifying subtle changes in ZFYVE9 localization. For endogenous biotin management, include avidin/streptavidin blocking steps before applying biotin-conjugated antibodies, and prepare control slides with streptavidin-conjugate only to assess background . Develop quantitative analysis pipelines using specialized software (CellProfiler, ImageJ) to measure parameters including mean fluorescence intensity, subcellular distribution ratios (membrane:cytoplasm, nuclear:cytoplasmic), and co-localization coefficients with endosomal markers or TGF-β pathway components. When analyzing co-localization, calculate both Pearson's and Mander's coefficients, reporting both values along with object-based co-localization metrics. For population heterogeneity assessment, perform single-cell analysis rather than relying solely on field averages, presenting data as frequency distributions to capture biological variability. Standardize region of interest (ROI) selection using automated algorithms based on cellular markers rather than subjective manual selection. Finally, develop integrated analysis workflows that combine immunofluorescence data with functional readouts (e.g., SMAD nuclear translocation, target gene expression) to correlate ZFYVE9 localization patterns with downstream signaling outcomes.

What are the emerging applications of ZFYVE9 antibodies in understanding disease mechanisms?

ZFYVE9 antibodies are increasingly valuable tools for investigating disease mechanisms, particularly in conditions where TGF-β signaling dysregulation plays a central role. In fibrotic disorders, ZFYVE9 antibodies can assess altered ZFYVE9-SMAD interactions that may contribute to excessive TGF-β signaling and fibrosis progression. For cancer research, these antibodies help investigate how ZFYVE9 expression or localization changes may contribute to the TGF-β paradox, where this pathway switches from tumor suppressive to tumor promoting. In developmental disorders, ZFYVE9 antibodies facilitate the study of embryonic developmental defects resulting from impaired TGF-β/SMAD signaling. Neurological disease studies benefit from ZFYVE9 antibodies to explore how aberrant endosomal trafficking (mediated by FYVE domain proteins) contributes to neurodegenerative conditions. For inflammatory disorders, these antibodies help elucidate how ZFYVE9, similar to ZFYVE1, might modulate innate immune responses through TLR-mediated pathways . Additionally, cardiovascular disease research uses ZFYVE9 antibodies to investigate endothelial-to-mesenchymal transition processes regulated by TGF-β. Future applications will likely expand to include therapeutic targeting validation, where ZFYVE9 antibodies will verify target engagement in drug development programs aimed at modulating TGF-β pathway activity through ZFYVE9 interactions.