ZFYVE9 Antibody, HRP conjugated

What is ZFYVE9 and why is it significant in cell signaling research?

ZFYVE9 (Zinc Finger FYVE Domain-Containing Protein 9) is an early endosomal protein that plays a critical role in TGF-β-mediated signaling pathways. It functions primarily to recruit SMAD2/SMAD3 to intracellular membranes and to the TGF-β receptor, thereby modulating SMAD3/SMAD4 complex transcriptional activity. The protein is also known by several other names including SARA (SMAD Anchor for Receptor Activation), MADHIP, NSP, PPP1R173, and SMADIP . ZFYVE9 is particularly significant in developmental biology, cancer research, and fibrosis studies due to its regulatory function in TGF-β signaling, which influences cell proliferation, differentiation, and apoptosis. Researchers investigate this protein to better understand signal transduction mechanisms and potential therapeutic targets in diseases involving aberrant TGF-β pathway activation.

What is the difference between unconjugated and HRP-conjugated ZFYVE9 antibodies?

Unconjugated ZFYVE9 antibodies consist solely of the immunoglobulin molecules that specifically recognize and bind to ZFYVE9 protein epitopes. These antibodies require a secondary detection system (typically a labeled secondary antibody) to visualize binding in experimental assays . In contrast, HRP-conjugated ZFYVE9 antibodies have horseradish peroxidase enzyme molecules directly attached to the antibody structure through covalent bonding . This conjugation eliminates the need for secondary antibodies in detection workflows, offering streamlined protocols with fewer steps and reduced background signal. The HRP enzyme catalyzes reactions with substrates like TMB or DAB to produce colorimetric, chemiluminescent, or fluorescent signals, enabling direct visualization and quantification of ZFYVE9 in various applications including Western blotting, ELISA, and immunohistochemistry.

How does the molecular weight of ZFYVE9 affect antibody selection and experimental design?

The observed molecular weight of ZFYVE9 is approximately 156kDa , which significantly impacts antibody selection and experimental design considerations. When selecting antibodies, researchers should verify that the antibody's epitope recognition corresponds to the full-length protein or specific domains of interest. The large molecular weight necessitates particular attention to protein extraction methods, as larger proteins can be more difficult to efficiently extract and transfer during Western blotting procedures. Experimental protocols may require extended transfer times, reduced gel percentages (typically 6-8% acrylamide), and specialized transfer buffers containing SDS or methanol to facilitate complete transfer of this high molecular weight protein. Additionally, researchers should be aware that post-translational modifications or alternative splicing may generate ZFYVE9 variants with different molecular weights, potentially resulting in multiple bands on Western blots, which requires careful validation with appropriate controls.

What are the optimal conditions for using HRP-conjugated ZFYVE9 antibodies in Western blotting?

For optimal Western blotting with HRP-conjugated ZFYVE9 antibodies, researchers should implement a comprehensive protocol considering the high molecular weight (156kDa) of the target protein . Begin by preparing protein samples in denaturing buffer with reducing agents, then separate using 6-8% SDS-PAGE gels to adequately resolve the large protein. For transfer, use PVDF membranes (rather than nitrocellulose) with extended transfer times (90-120 minutes) at lower voltages or overnight transfers at 4°C to ensure complete transfer of this large protein.

When blocking, a 5% non-fat dry milk or BSA solution in TBST is typically effective. For the primary antibody incubation, dilute the HRP-conjugated ZFYVE9 antibody to the manufacturer's recommended range (typically 1:500-1:2000 for Western blotting) , and incubate for 1-2 hours at room temperature or overnight at 4°C. After thorough washing with TBST buffer (4-5 washes of 5-10 minutes each), proceed directly to detection using enhanced chemiluminescence (ECL) substrate—no secondary antibody is needed as the HRP is already conjugated to the primary antibody. Positive controls such as LO2 or HeLa cell lysates known to express ZFYVE9 should be included to validate signal specificity .

How should researchers optimize immunofluorescence protocols using ZFYVE9 antibodies?

Optimizing immunofluorescence (IF) protocols with ZFYVE9 antibodies requires careful attention to fixation, permeabilization, and antibody dilution factors. For cultured cells, begin with 4% paraformaldehyde fixation for 15-20 minutes at room temperature, followed by permeabilization with 0.1-0.3% Triton X-100 for 10 minutes to ensure antibody access to intracellular ZFYVE9. Blocking with 5-10% normal serum (from the species in which the secondary antibody was raised) for 30-60 minutes reduces non-specific binding.

For unconjugated primary ZFYVE9 antibodies, dilutions between 1:50 and 1:200 are recommended for IF applications . When using HRP-conjugated ZFYVE9 antibodies for IF, an additional tyramide signal amplification (TSA) step is necessary to convert the HRP enzymatic activity into a fluorescent signal. Incubation times should be optimized through titration experiments, typically starting with 1-2 hours at room temperature or overnight at 4°C. Include nuclear counterstains (e.g., DAPI) and consider co-staining with early endosomal markers (e.g., EEA1) to confirm the expected subcellular localization of ZFYVE9. For particularly challenging samples, antigen retrieval methods may be necessary, especially in tissue sections where epitope accessibility might be limited.

What are the key considerations when conjugating ZFYVE9 antibodies with HRP in the laboratory?

When performing in-house HRP conjugation of ZFYVE9 antibodies, several critical factors must be considered to ensure successful conjugation while maintaining antibody functionality. First, antibody purity is paramount—use affinity-purified antibodies in appropriate buffer conditions (10-50mM amine-free buffers like HEPES, MES, MOPS, or phosphate at pH 6.5-8.5) . Avoid buffers containing nucleophilic components such as primary amines and thiols, as these may interfere with the conjugation chemistry. Sodium azide, a common antibody preservative, must be completely removed through dialysis or desalting as it irreversibly inhibits HRP activity .

For optimal conjugation using commercial kits like the LYNX Rapid HRP Antibody Conjugation Kit, the molar ratio between antibody and HRP should ideally be between 1:1 and 1:4 (antibody to HRP). Given the molecular weights (approximately 160,000 for antibodies versus 40,000 for HRP), this translates to using 5-20mg of antibody for 5mg of HRP . The antibody concentration should range from 0.5-5.0mg/ml in a volume up to 5ml for optimal results . After conjugation, validate the conjugate's activity through functional assays such as ELISA or Western blotting, comparing performance against commercial conjugates or unconjugated primary antibody plus HRP-labeled secondary antibody systems.

How can researchers address non-specific binding when using HRP-conjugated ZFYVE9 antibodies?

Non-specific binding is a common challenge with HRP-conjugated ZFYVE9 antibodies that can compromise experimental results. To systematically address this issue, first optimize blocking conditions by testing different blocking agents (5% BSA, 5% non-fat dry milk, commercial blocking buffers) and extending blocking times to 1-2 hours at room temperature. Dilution optimization is equally important—begin with the manufacturer's recommended range (1:500-1:2000 for Western blots, 1:50-1:200 for IF) and perform titration experiments to identify the optimal concentration that maximizes specific signal while minimizing background.

Including appropriate controls is essential for distinguishing between true and false signals. These should include: (1) a negative control omitting the primary antibody, (2) a peptide competition assay where the antibody is pre-incubated with the immunizing peptide, and (3) positive controls using cell lines known to express ZFYVE9 (LO2 or HeLa cells) . If non-specific binding persists, consider more stringent washing conditions (increased salt concentration or mild detergents in wash buffers) and shorter substrate incubation times to minimize background development. For particularly problematic samples, pre-absorption of the antibody with proteins from the species being tested or with cell/tissue lysates lacking the target protein can significantly reduce cross-reactivity.

What factors might lead to inconsistent results in ZFYVE9 detection across different experimental systems?

Inconsistent ZFYVE9 detection across experimental systems can stem from multiple factors that require systematic investigation. Epitope accessibility varies significantly between applications—the ZFYVE9 antibody recognition site may be exposed differently in denaturing Western blots versus native-state immunofluorescence. Different antibodies targeting distinct epitopes (e.g., AA 1-260, AA 1-762, AA 71-170, AA 683-926) may perform differently depending on protein conformation and post-translational modifications.

Expression level variability also contributes to inconsistency—ZFYVE9 expression can differ dramatically between cell types, tissues, and experimental conditions, particularly in response to TGF-β pathway activation. Technical factors including protein extraction methods, buffer compositions, fixation protocols, and incubation conditions must be standardized to ensure reproducibility. For HRP-conjugated antibodies specifically, enzyme activity can be compromised by improper storage, freeze-thaw cycles, or exposure to inhibitors like sodium azide .

To address these inconsistencies, researchers should: (1) validate antibodies across multiple applications before extensive use, (2) use consistent positive controls in every experiment, (3) standardize protocols including buffer compositions and incubation times, and (4) consider using multiple antibodies targeting different ZFYVE9 epitopes to confirm results, especially in novel experimental systems.

How should researchers interpret multiple bands when detecting ZFYVE9 in Western blotting?

When Western blot analysis of ZFYVE9 reveals multiple bands rather than the expected single band at 156kDa , systematic interpretation is required to distinguish between legitimate protein variants and technical artifacts. First, consider the biological explanations: ZFYVE9 may undergo alternative splicing generating multiple isoforms, post-translational modifications (phosphorylation, ubiquitination, SUMOylation) that alter molecular weight, or proteolytic processing creating truncated forms with functional significance. The specific antibody epitope location significantly impacts which forms are detectable—antibodies targeting different regions (N-terminal AA 1-260 versus C-terminal AA 683-926) might recognize different subsets of protein variants.

Technical explanations for multiple bands include incomplete denaturation leading to different migration patterns, non-specific binding to related proteins containing similar domains (particularly other FYVE domain-containing proteins), protein degradation during sample preparation, or incomplete transfer of the full-length 156kDa protein. To properly interpret these patterns, researchers should implement validation strategies including: (1) comparing results with multiple ZFYVE9 antibodies targeting different epitopes, (2) performing RNA interference experiments to confirm which bands diminish with ZFYVE9 knockdown, (3) using recombinant ZFYVE9 protein as a positive control for size verification, and (4) analyzing ZFYVE9 expression in various cell lines with known expression patterns as comparative references.

How can HRP-conjugated ZFYVE9 antibodies be optimized for quantitative analysis of TGF-β signaling dynamics?

Optimizing HRP-conjugated ZFYVE9 antibodies for quantitative analysis of TGF-β signaling dynamics requires a sophisticated approach integrating multiple experimental systems. Begin by establishing standard curves using recombinant ZFYVE9 protein at known concentrations to define the linear detection range of your HRP-conjugated antibody system. For time-course experiments monitoring ZFYVE9's role in TGF-β signaling, synchronize cells through serum starvation (6-24 hours) before stimulation with precise TGF-β concentrations (typically 1-10 ng/ml), then collect samples at defined intervals (5, 15, 30, 60, 120 minutes and longer timepoints) to capture both rapid and delayed responses.

Quantitative Western blotting requires careful normalization strategies—normalize ZFYVE9 signals to stable housekeeping proteins and include internal calibration standards on each blot. For more precise quantification, consider ELISA-based approaches using the HRP-conjugated ZFYVE9 antibody in a sandwich format with a capture antibody targeting a different ZFYVE9 epitope. Digital image analysis of immunofluorescence data can provide spatial information about ZFYVE9 translocation during TGF-β signaling—measure colocalization coefficients between ZFYVE9 and markers for different subcellular compartments (early endosomes, plasma membrane, cytoplasm) using software like ImageJ with colocalization plugins.

For systems-level analysis, correlate ZFYVE9 dynamics with downstream SMAD2/3 phosphorylation, nuclear translocation, and target gene expression to create comprehensive signaling profiles. This integrated approach allows researchers to quantitatively define how ZFYVE9 coordinates TGF-β signal transduction across different cellular contexts and experimental conditions.

What are the considerations for using ZFYVE9 antibodies in multiplex immunoassays with other TGF-β pathway components?

Developing multiplex immunoassays incorporating ZFYVE9 antibodies alongside other TGF-β pathway components requires careful attention to antibody compatibility, signal separation, and validation strategies. When selecting antibody panels, choose ZFYVE9 antibodies and antibodies against other pathway components (TGF-β receptors, SMAD2/3, SMAD4, SMURF proteins) from different host species to prevent cross-reactivity in detection systems. If using multiple rabbit-derived antibodies, consider sequential staining protocols with complete stripping between rounds or employ antibodies directly labeled with distinct fluorophores or enzymes.

For HRP-conjugated ZFYVE9 antibodies in chromogenic multiplex systems, tyramide signal amplification (TSA) with different fluorophores allows sequential detection while maintaining sensitivity. Optimize antibody dilutions individually before combining in multiplex format, as optimal concentrations may differ from single-plex applications. Potential antibody interference must be experimentally evaluated—pre-test combinations to ensure one antibody doesn't sterically hinder another's binding when targets are in close proximity, particularly for protein complexes formed during active TGF-β signaling.

Rigorous validation for multiplex systems includes single-color controls (each antibody tested alone), absorption controls (pre-incubation with immunizing peptides), and biological validation comparing staining patterns in cells with normal versus disrupted TGF-β signaling (through receptor inhibitors or SMAD knockdowns). Quantitative analysis of multiplex data should include colocalization measurements between ZFYVE9 and other pathway components, providing insights into protein-protein interactions during signal transduction that cannot be obtained from single-target approaches.

How can researchers effectively use ZFYVE9 antibodies to investigate its role in endosomal trafficking and receptor internalization?

Investigating ZFYVE9's role in endosomal trafficking and receptor internalization requires sophisticated experimental approaches centered around appropriate antibody applications. For live-cell imaging studies, convert the HRP-conjugated ZFYVE9 antibody signal into fluorescence using tyramide signal amplification in fixed timepoint experiments, or consider using unconjugated ZFYVE9 antibodies directly labeled with fluorophores for compatible live-cell applications. Pulse-chase experiments can track TGF-β receptor internalization and colocalization with ZFYVE9—label cell-surface TGF-β receptors with biotin or fluorescent antibodies, stimulate with ligand, then fix at defined timepoints to assess colocalization with ZFYVE9 in different endosomal compartments.

Super-resolution microscopy techniques (STORM, PALM, STED) provide nanoscale resolution of ZFYVE9's distribution in endosomal structures, requiring careful antibody validation at these higher resolutions. For biochemical approaches, use HRP-conjugated ZFYVE9 antibodies in immunoprecipitation followed by mass spectrometry to identify ZFYVE9-associated proteins in endosomal compartments under different signaling conditions. Subcellular fractionation with differential centrifugation to isolate endosomal compartments, followed by Western blotting with HRP-conjugated ZFYVE9 antibodies, can quantify ZFYVE9 distribution across cellular compartments.

Functional studies can employ proximity ligation assays (PLA) to visualize and quantify interactions between ZFYVE9 and TGF-β receptors or other endosomal proteins with single-molecule sensitivity. For mechanistic insights, combine these approaches with genetic manipulation (CRISPR/Cas9 editing of ZFYVE9 FYVE domain or protein-binding regions) and assess the impact on receptor trafficking using the established imaging and biochemical assays, creating a comprehensive understanding of ZFYVE9's role in coordinating endosomal trafficking events in TGF-β signaling.

What positive and negative controls should be included when validating HRP-conjugated ZFYVE9 antibodies?

A comprehensive validation strategy for HRP-conjugated ZFYVE9 antibodies requires carefully selected positive and negative controls. For positive controls, include cell lines with confirmed ZFYVE9 expression such as HeLa and LO2 cells, which have been documented as suitable positive controls . Recombinant ZFYVE9 protein or overexpression systems (cells transfected with ZFYVE9 expression vectors) provide additional positive controls with defined expression levels. TGF-β stimulated versus unstimulated cells serve as physiologically relevant positive controls, as ZFYVE9 function is closely linked to TGF-β signaling activation .

Negative controls should include: (1) isotype controls using non-specific IgG of the same host species, concentration, and conjugation status as the ZFYVE9 antibody; (2) peptide competition/neutralization controls where the antibody is pre-incubated with excess immunizing peptide to block specific binding sites; (3) ZFYVE9 knockdown or knockout systems created using siRNA or CRISPR/Cas9 to specifically reduce or eliminate ZFYVE9 expression; and (4) cell lines with minimal ZFYVE9 expression (determined through literature review or preliminary screening).

For HRP conjugates specifically, include additional controls to verify enzyme activity: (1) unconjugated primary ZFYVE9 antibody plus HRP-labeled secondary antibody as a reference for normal signal, (2) substrate-only controls to assess background peroxidase activity, and (3) non-specific HRP-conjugated antibodies to distinguish between specific signal and potential artifacts from the HRP conjugation process itself.

How do buffer compositions and storage conditions affect HRP-conjugated ZFYVE9 antibody performance?

The presence of 50% glycerol serves as a cryoprotectant preventing freeze-thaw damage, while BSA provides stability through preventing non-specific binding to storage container surfaces. Buffer pH significantly impacts both antibody binding affinity and HRP enzymatic activity—pH values below 6.0 or above 8.5 can compromise performance. For working dilutions, avoid buffers containing primary amines (such as high concentrations of Tris) or thiols as these can interfere with the chemistry of certain HRP substrates .

Storage temperature recommendations include keeping HRP-conjugated ZFYVE9 antibodies at -20°C for long-term storage , avoiding repeated freeze-thaw cycles by preparing single-use aliquots, and storing working dilutions at 4°C for no more than 1-2 weeks. The combined chemical and thermal stability profile of these conjugates necessitates careful handling—exposure to oxidizing agents, heavy metals, or extreme pH changes should be avoided as these can irreversibly damage HRP activity or antibody structure. Periodic validation of aged conjugates through positive control testing is recommended to ensure continued performance over time.

What methodological approaches can distinguish between specific ZFYVE9 isoforms or post-translationally modified variants?

Distinguishing between specific ZFYVE9 isoforms or post-translationally modified variants requires sophisticated methodological approaches centered around strategic antibody selection and complementary techniques. Epitope-specific antibody selection is foundational—choose antibodies recognizing distinct regions of ZFYVE9 (N-terminal, central domains, C-terminal) to identify potential isoforms. The available antibodies targeting amino acids 1-260, 71-170, 1-762, and 683-926 can be strategically employed to create a "mapping" of detected variants, with differential detection patterns revealing structural variations.

For post-translational modifications (PTMs), combine general ZFYVE9 antibodies with PTM-specific approaches. These include: (1) treatment with specific enzymes (phosphatases, deubiquitinases, or deglycosylases) before Western blotting to identify which bands shift after modification removal; (2) immunoprecipitation with ZFYVE9 antibodies followed by Western blotting with modification-specific antibodies (anti-phospho, anti-ubiquitin, anti-SUMO); and (3) mass spectrometry analysis of immunoprecipitated ZFYVE9 to precisely identify and locate PTMs.

Two-dimensional gel electrophoresis provides powerful resolution of protein variants—separate ZFYVE9 first by isoelectric point (affected by phosphorylation and other charged modifications) and then by molecular weight, followed by Western blotting with HRP-conjugated ZFYVE9 antibodies. This creates characteristic spot patterns for different ZFYVE9 variants. RT-PCR and sequencing approaches complement protein-level analyses by identifying alternative splicing events that generate distinct isoforms, allowing correlation between detected protein variants and their encoding transcripts. Computational prediction tools can guide experimental design by identifying likely PTM sites or alternative splice junctions for targeted investigation.