ZFYVE9 Antibody, FITC conjugated

What is ZFYVE9 protein and what are its key cellular functions?

ZFYVE9 (Zinc finger FYVE domain-containing protein 9) is an early endosomal protein that plays a crucial role in TGF-β signaling pathways. It functions primarily to recruit SMAD2/SMAD3 to intracellular membranes and to the TGF-beta receptor. The protein significantly contributes to TGF-mediated signaling by regulating the subcellular localization of SMAD2 and SMAD3 and modulating the transcriptional activity of the SMAD3/SMAD4 complex. ZFYVE9 is also potentially associated with TGF-beta receptor internalization processes .

The protein contains multiple functional domains, including a FYVE-type zinc finger that is necessary and sufficient for its localization to early endosomes through association with phosphatidylinositol 3-phosphate (PI3P). Additionally, ZFYVE9 contains a SMAD binding domain (SBD) that interacts with the MH2 domains of SMAD2 or SMAD3, facilitating their recruitment to receptor complexes .

What are the structural characteristics of FITC-conjugated ZFYVE9 antibodies?

FITC-conjugated ZFYVE9 antibodies, such as ABIN7176070, are typically polyclonal antibodies raised in rabbits against specific amino acid sequences of human ZFYVE9. The particular antibody referenced in the search results targets amino acids 683-926 of human ZFYVE9 protein. The antibody is purified through antigen affinity methods to ensure specificity and then conjugated to fluorescein isothiocyanate (FITC), enabling direct fluorescent detection in various applications .

The FITC conjugation eliminates the need for secondary antibody incubation steps in fluorescence-based applications, streamlining experimental workflows. These antibodies are typically supplied in liquid form, preserved in buffers containing components like ProClin 300 (0.03%), glycerol (50%), and PBS (pH 7.4) to maintain stability and functionality during storage and use .

What experimental applications are suitable for FITC-conjugated ZFYVE9 antibodies?

FITC-conjugated ZFYVE9 antibodies are versatile research tools applicable to multiple experimental techniques. While specific applications may vary between products, common applications include:

• Immunofluorescence (IF) - For visualization of ZFYVE9 localization in fixed cells and tissues

• Flow cytometry (FACS) - For quantitative analysis of ZFYVE9 expression in cell populations

• ELISA - For quantitative detection of ZFYVE9 in various sample types

For unconjugated versions of ZFYVE9 antibodies, additional applications such as Western blotting (WB), immunoprecipitation (IP), and immunohistochemistry (IHC) have been documented. The optimal dilution ranges vary by application, with typical ranges of 1:50-1:200 for immunofluorescence and 1:200-1:2000 for Western blot applications .

Researchers should note that experimental conditions may require optimization for each specific research context, and manufacturer recommendations should be consulted for initial protocol development.

What are optimal sample preparation methods for ZFYVE9 immunofluorescence studies?

When designing immunofluorescence experiments with FITC-conjugated ZFYVE9 antibodies, proper sample preparation is critical for obtaining specific and reliable results. Since ZFYVE9 is primarily localized to early endosomes, protocols should preserve endosomal structures while ensuring antibody accessibility.

Recommended cell fixation protocol:

• Wash cells gently with pre-warmed PBS (37°C) twice

• Fix cells with 4% paraformaldehyde in PBS for 15-20 minutes at room temperature

• Permeabilize with 0.1-0.2% Triton X-100 in PBS for 5-10 minutes

Alternative fixation methods:

• For better preservation of membrane structures, consider using methanol fixation (-20°C for 10 minutes)

• For double labeling with other endosomal markers, optimize fixation conditions to accommodate all antibodies

When working with the FITC-conjugated ZFYVE9 antibody, researchers should be aware that FITC fluorescence is pH-sensitive and photobleaches relatively quickly. Therefore, using mounting media with anti-fade agents and pH stabilizers is recommended to preserve signal during imaging and storage .

How should researchers validate specificity of ZFYVE9 antibodies in their experimental systems?

Antibody validation is essential for ensuring reliable and reproducible results. For ZFYVE9 antibodies, consider implementing these validation strategies:

Table 1: Validation Methods for ZFYVE9 Antibody Specificity

Additionally, researchers should consider co-localization studies with established early endosomal markers (e.g., EEA1) to confirm the expected subcellular distribution pattern of ZFYVE9. The antibody should exhibit a punctate staining pattern consistent with endosomal structures .

What technical considerations are important when optimizing FITC-conjugated antibody dilutions?

Determining the optimal antibody concentration is crucial for maximizing specific signal while minimizing background. For FITC-conjugated ZFYVE9 antibodies, consider this systematic approach:

• Start with the manufacturer's recommended dilution range (e.g., 1:50-1:200 for IF applications)

• Perform a titration experiment using 3-4 different dilutions across this range

• Include appropriate positive and negative controls

• Evaluate signal-to-noise ratio at each dilution

• Select the dilution that provides maximal specific signal with minimal background

Important technical considerations:

• FITC has an excitation maximum at approximately 495 nm and emission maximum around 519 nm

• Avoid prolonged exposure to light during all experimental steps to prevent photobleaching

• Be aware that some fixatives can increase cellular autofluorescence in the FITC channel

• Consider counterstaining with DAPI to visualize nuclei while minimizing spectral overlap

• When using multiple fluorophores, ensure appropriate filter sets to avoid bleed-through

For quantitative applications, preparation of a standard curve using recombinant ZFYVE9 protein can help establish the linear detection range of the antibody at the selected dilution .

What are common causes of high background when using FITC-conjugated ZFYVE9 antibodies?

High background is a frequent challenge when working with fluorescently labeled antibodies. For FITC-conjugated ZFYVE9 antibodies, several factors may contribute to elevated background:

Common causes and solutions for high background:

• Insufficient blocking:

  • Increase blocking time (1-2 hours)

  • Try alternative blocking agents (5% BSA, 5-10% normal serum, commercial blocking buffers)

• Excessive antibody concentration:

  • Dilute antibody further based on titration experiments

  • Reduce incubation time if using higher concentrations

• Inadequate washing:

  • Increase number of wash steps (5-6 washes)

  • Extend washing time (10 minutes per wash)

  • Use gentle agitation during washing

• Fixation-induced autofluorescence:

  • Reduce fixation time

  • Treat samples with autofluorescence reducers (e.g., sodium borohydride)

  • Consider alternative fixation methods

• Nonspecific binding:

  • Add 0.1-0.3% Triton X-100 to antibody dilution buffer

  • Include 0.05-0.1% Tween-20 in wash buffers

Implementing these solutions systematically can help identify and address the specific source of background in your experimental system, leading to cleaner and more interpretable results .

How can researchers address weak or absent signal when using ZFYVE9 antibodies?

Weak or absent signals can result from various factors when working with ZFYVE9 antibodies. The following troubleshooting approach can help identify and resolve these issues:

Table 2: Troubleshooting Weak ZFYVE9 Antibody Signals

Additionally, consider that ZFYVE9 expression may be stimulus-dependent. Treatment with TGF-β may enhance detection by increasing ZFYVE9 recruitment to endosomal compartments. When working with tissue samples, antigen retrieval methods may be necessary to expose the epitope recognized by the antibody .

What strategies can address potential cross-reactivity issues with ZFYVE9 antibodies?

Cross-reactivity can compromise the reliability of experimental results when using antibodies. For ZFYVE9 antibodies, consider implementing these strategies to address potential cross-reactivity:

• Species considerations:

  • Confirm the species reactivity of your antibody matches your experimental system

  • Note that some ZFYVE9 antibodies are specifically human-reactive, while others cross-react with mouse and rat

• Validation in knockout/knockdown systems:

  • Use CRISPR/Cas9-generated ZFYVE9 knockout cells as negative controls

  • Compare staining patterns in ZFYVE9 siRNA-treated vs. control cells

• Epitope analysis:

  • Check for sequence homology between the immunizing peptide (e.g., AA 683-926) and other proteins

  • Consider using antibodies targeting different epitopes of ZFYVE9 for confirmation

• Absorption controls:

  • Pre-absorb antibody with the immunizing peptide before application

  • A significant signal reduction indicates specific binding

• Western blot verification:

  • Confirm antibody detects a single band at the expected molecular weight (approximately 156 kDa)

  • Multiple bands may indicate cross-reactivity with other proteins

By implementing these approaches, researchers can increase confidence in the specificity of their ZFYVE9 antibody and minimize misinterpretation of results due to cross-reactivity .

How can ZFYVE9 antibodies be employed in co-localization studies with endosomal markers?

Co-localization studies can provide valuable insights into ZFYVE9 function within the endosomal system. Here's a methodological approach for conducting such studies:

• Selection of appropriate endosomal markers:

  • Early endosomes: EEA1, Rab5

  • Recycling endosomes: Rab11

  • Late endosomes: Rab7

  • Multivesicular bodies: CD63

• Immunofluorescence co-staining protocol:

  • Fix cells using 4% paraformaldehyde (10 minutes)

  • Permeabilize with 0.2% Triton X-100 (5 minutes)

  • Block with 5% normal serum (1 hour)

  • Co-incubate with FITC-conjugated ZFYVE9 antibody and a different fluorophore-conjugated endosomal marker (or unconjugated primary followed by fluorescently-labeled secondary)

  • Counterstain nuclei with DAPI

  • Mount with anti-fade medium

• Confocal microscopy acquisition:

  • Use sequential scanning to minimize bleed-through

  • Optimize pinhole settings for optimal z-resolution

  • Capture multiple fields to ensure representative sampling

• Quantitative co-localization analysis:

  • Calculate Pearson's or Mander's coefficients

  • Perform line scan analysis across endosomal structures

  • Consider 3D reconstruction for volumetric co-localization assessment

Such studies can reveal whether ZFYVE9 predominantly localizes to specific endosomal compartments and how this localization may change upon TGF-β stimulation or other experimental conditions .

What approaches can be used to study the dynamics of ZFYVE9 in TGF-β receptor internalization?

ZFYVE9's role in TGF-β receptor internalization can be studied using several advanced approaches:

Pulse-chase receptor internalization assay:

• Surface-label TGF-β receptors with biotin or a non-permeabilizing antibody approach

• Stimulate with TGF-β to induce internalization

• Fix cells at different time points (0, 5, 15, 30, 60 minutes)

• Stain with FITC-conjugated ZFYVE9 antibody

• Quantify co-localization between internalized receptors and ZFYVE9

Live-cell imaging approach:

• Generate cells expressing fluorescently-tagged TGF-β receptor

• Transiently transfect with a spectrally distinct fluorescent ZFYVE9 construct

• Perform time-lapse imaging before and after TGF-β stimulation

• Analyze temporal dynamics of receptor-ZFYVE9 interactions

Biochemical fractionation approach:

• Stimulate cells with TGF-β for various durations

• Isolate endosomal fractions using gradient centrifugation

• Analyze fractions by Western blotting for ZFYVE9 and TGF-β receptor

• Quantify the kinetics of receptor-ZFYVE9 association in endosomal compartments

These approaches can provide complementary insights into how ZFYVE9 participates in receptor trafficking and signaling, potentially revealing new aspects of TGF-β pathway regulation .

How can researchers use ZFYVE9 antibodies to investigate SMAD2/3 recruitment and signaling dynamics?

The role of ZFYVE9 in recruiting SMAD2/3 to TGF-β receptors makes it a key protein for understanding signaling initiation. Here are methodological approaches to study this process:

• Proximity ligation assay (PLA):

  • Fix cells at different time points after TGF-β stimulation

  • Incubate with ZFYVE9 antibody and anti-SMAD2/3 antibody

  • Perform PLA according to manufacturer's protocol

  • Quantify PLA spots to measure temporal dynamics of interaction

• Immunoprecipitation-based approach:

  • Stimulate cells with TGF-β for various durations

  • Immunoprecipitate with ZFYVE9 antibody

  • Analyze precipitates for co-immunoprecipitated SMAD2/3 and TGF-β receptor

  • Western blot for phosphorylated vs. total SMAD2/3

• Fluorescence resonance energy transfer (FRET):

  • Express ZFYVE9-CFP and SMAD2/3-YFP fusion proteins

  • Measure FRET efficiency before and after TGF-β stimulation

  • Calculate interaction dynamics in living cells

• ZFYVE9 domain mutation analysis:

  • Generate cells expressing ZFYVE9 with mutations in the SMAD binding domain

  • Assess SMAD2/3 phosphorylation, nuclear translocation, and target gene expression

  • Use FITC-conjugated wild-type ZFYVE9 antibody to compare localization patterns

These approaches can provide detailed insights into how ZFYVE9 coordinates the early events in TGF-β signaling and how disruption of this process might affect downstream signaling outcomes in various biological contexts .

What quantitative approaches can be used to analyze ZFYVE9 immunofluorescence data?

Quantitative analysis of ZFYVE9 immunofluorescence provides objective metrics for comparing experimental conditions. Researchers can employ these analytical approaches:

• Intensity-based measurements:

  • Mean fluorescence intensity (MFI) within defined cellular regions

  • Integrated density (product of area and mean intensity)

  • Background-corrected total cell fluorescence (BCTCF)

• Pattern analysis:

  • Count of ZFYVE9-positive puncta per cell

  • Size distribution of ZFYVE9-positive structures

  • Nearest neighbor distance between puncta

• Subcellular distribution:

  • Nuclear:cytoplasmic intensity ratio

  • Perinuclear:peripheral intensity ratio

  • Distance of puncta from plasma membrane or nuclear envelope

• Co-localization metrics:

  • Pearson's correlation coefficient with endosomal markers

  • Mander's overlap coefficient for partial co-localization

  • Object-based co-localization for discrete structures

Table 3: Example Quantification Workflow for ZFYVE9 Immunofluorescence

How should researchers interpret changes in ZFYVE9 distribution following TGF-β stimulation?

Changes in ZFYVE9 distribution following TGF-β stimulation can provide insights into signaling mechanisms. Here's a guide to interpreting these changes:

• Expected response patterns:

  • Increased co-localization with internalized TGF-β receptors

  • Enhanced punctate staining pattern consistent with endosomal recruitment

  • Potential changes in endosomal size or distribution

• Temporal considerations:

  • Early response (5-15 minutes): Initial recruitment to receptors

  • Intermediate response (15-30 minutes): Peak of SMAD2/3 recruitment

  • Late response (30-120 minutes): Possible receptor recycling or degradation phases

• Context-dependent variations:

  • Cell type-specific differences in magnitude or kinetics

  • Impact of cell density and polarization on response patterns

  • Influence of other signaling pathways on ZFYVE9 dynamics

• Functional correlations:

  • Relationship between ZFYVE9 redistribution and SMAD2/3 phosphorylation

  • Association between aberrant ZFYVE9 patterns and altered gene expression

  • Correlation between ZFYVE9 dynamics and biological outcomes

When interpreting these changes, researchers should consider that ZFYVE9's role extends beyond simply recruiting SMADs to receptors; it may also influence the balance between different receptor trafficking routes, potentially affecting signal duration and intensity .

What considerations are important when correlating ZFYVE9 localization with disease models or genetic variations?

When investigating ZFYVE9 in disease contexts or in relation to genetic variations, several analytical considerations are important:

• Disease-specific changes:

  • Altered expression levels (quantified by intensity measurements)

  • Abnormal subcellular distribution patterns

  • Changed co-localization with pathway components

  • Modified dynamic responses to TGF-β stimulation

• Genetic variation impact:

  • Effects of ZFYVE9 mutations on protein localization and function

  • Influence of IgG1 allotypic markers (G1m) on antibody responses (relevant in immunological studies)

  • Correlation between genotype and ZFYVE9 expression or localization patterns

• Analytical approaches:

  • Stratify samples by genetic markers or disease status

  • Perform blinded quantitative analysis to avoid bias

  • Use machine learning for pattern recognition in complex datasets

  • Consider multiplexed approaches to correlate ZFYVE9 with other disease markers

• Interpretation guidelines:

  • Distinguish between causative changes and secondary adaptations

  • Consider cell type-specific roles in disease progression

  • Assess whether alterations affect specific protein domains with distinct functions

  • Evaluate conservation of findings across multiple model systems

These considerations can help researchers establish meaningful connections between ZFYVE9 biology and disease processes, potentially identifying new therapeutic targets or diagnostic markers .

What are the detailed specifications of commercially available FITC-conjugated ZFYVE9 antibodies?

When selecting a FITC-conjugated ZFYVE9 antibody for research applications, understanding the detailed specifications is essential. Based on the available search results, here are the key specifications for a representative product:

Table 4: Technical Specifications for FITC-Conjugated ZFYVE9 Antibody

The antibody targets a specific region of ZFYVE9 that may be important for its function or particularly immunogenic. The polyclonal nature suggests recognition of multiple epitopes within the target region, which may provide robust detection but potentially increased background compared to monoclonal alternatives .

How do researchers determine the optimal storage conditions for preserving FITC conjugate activity?

Maintaining the activity of FITC-conjugated antibodies requires careful attention to storage conditions. The following guidelines help preserve both antibody functionality and fluorophore activity:

• Temperature considerations:

  • Store at -20°C for long-term preservation

  • Working aliquots can be kept at 4°C for up to one month

  • Avoid storing at room temperature for extended periods

• Light protection:

  • Store in amber vials or wrap containers in aluminum foil

  • Minimize exposure to light during all handling steps

  • Consider light-blocking storage boxes

• Aliquoting strategy:

  • Prepare small single-use aliquots to avoid repeated freeze-thaw cycles

  • Typical aliquot volumes of 10-25 μL minimize waste

  • Include date of aliquoting and thawing on labels

• Buffer optimizations:

  • The presence of 50% glycerol helps prevent freezing damage

  • Preservatives like ProClin 300 (0.03%) inhibit microbial growth

  • Neutral pH (7.4) helps maintain FITC fluorescence properties

• Stability monitoring:

  • Periodically test aliquots on positive control samples

  • Monitor for decreased fluorescence intensity over time

  • Check for increased background or non-specific binding

By following these guidelines, researchers can maximize the useful lifespan of FITC-conjugated ZFYVE9 antibodies, ensuring consistent performance across experiments and reducing variability in results .

How might ZFYVE9 antibodies contribute to understanding TGF-β pathway dysregulation in disease?

ZFYVE9 antibodies represent valuable tools for investigating TGF-β pathway dysregulation in various pathological conditions. Potential research applications include:

• Cancer research applications:

  • Examining ZFYVE9 expression and localization in tumor versus normal tissues

  • Investigating correlation between ZFYVE9 alterations and TGF-β's dual role in tumor suppression/promotion

  • Studying how ZFYVE9 dynamics influence epithelial-mesenchymal transition (EMT)

• Fibrotic disease investigations:

  • Analyzing ZFYVE9 expression patterns in fibrotic tissues

  • Correlating ZFYVE9 localization with myofibroblast activation

  • Determining whether ZFYVE9 targeting could modulate excessive TGF-β signaling

• Immune regulation studies:

  • Examining ZFYVE9's role in TGF-β-mediated immune suppression

  • Investigating potential connections to vaccine response variability

  • Studying impact of ZFYVE9 on regulatory T cell development and function

• Neurodegenerative disease research:

  • Analyzing ZFYVE9 expression in reactive astrocytes and cerebrovascular smooth muscle cells

  • Investigating potential roles in neuroinflammatory processes

  • Studying connections to protein aggregation pathways

These research directions could potentially identify ZFYVE9 as a therapeutic target or biomarker in conditions characterized by aberrant TGF-β signaling, opening new avenues for diagnostic and treatment approaches .

What novel methodological approaches might enhance ZFYVE9 detection and functional analysis?

Emerging technologies and methodological innovations could enhance our ability to study ZFYVE9 biology:

• Advanced imaging approaches:

  • Super-resolution microscopy (STORM, PALM, SIM) for nanoscale localization

  • Light-sheet microscopy for 3D tissue analysis with minimal photobleaching

  • Expansion microscopy for physical magnification of subcellular structures

• Multiplexed detection systems:

  • Imaging mass cytometry for simultaneous detection of dozens of proteins

  • Sequential immunofluorescence for comprehensive pathway mapping

  • Multiplexed ion beam imaging (MIBI) for high-parameter tissue analysis

• Live-cell functional assays:

  • CRISPR-based endogenous tagging of ZFYVE9 with fluorescent proteins

  • Optogenetic control of ZFYVE9 localization or interactions

  • FRET-based biosensors for monitoring ZFYVE9-protein interactions in real-time

• Single-cell approaches:

  • Combining ZFYVE9 protein detection with single-cell transcriptomics

  • Studying cell-to-cell variability in ZFYVE9 expression and localization

  • Correlating ZFYVE9 status with cellular phenotypes at single-cell resolution