ZP2 Antibody, FITC conjugated

What is ZP2 and why is it important in reproductive research?

ZP2 is a glycoprotein component of the zona pellucida, an extracellular matrix surrounding oocytes. It functions as a secondary sperm receptor, binding to sperm after the acrosome reaction has been induced. This interaction facilitates the initial binding of sperm to the oocyte, which is essential for successful fertilization. During fertilization, ZP2 undergoes proteolytic cleavage, altering its function. The dynamic changes in ZP2 and its interactions with other zona pellucida proteins (ZP1, ZP3, and ZP4) highlight its importance in reproductive biology research and fertility studies .

How does ZP2 Antibody, FITC conjugated differ from other ZP2 antibody formats?

ZP2 Antibody with FITC (fluorescein isothiocyanate) conjugation provides direct fluorescent visualization capabilities not available with unconjugated antibodies. Unlike agarose or HRP conjugates that require additional steps or substrates for detection, FITC-conjugated antibodies emit green fluorescence (excitation ~495 nm, emission ~519 nm) when exposed to appropriate wavelength light. This enables direct visualization in applications like immunofluorescence microscopy and flow cytometry without secondary antibody requirements. The C-7 mouse monoclonal IgG1 kappa light chain version available commercially detects ZP2 of human origin and has validated applications in western blotting, immunoprecipitation, immunofluorescence, and ELISA .

What are the validated research applications for ZP2 Antibody, FITC conjugated?

ZP2 Antibody with FITC conjugation has been validated for:

• Immunofluorescence (IF) - Direct visualization of ZP2 localization in oocytes, embryos, and reproductive tissues

• Flow cytometry - Analysis of ZP2 expression in single-cell suspensions

• Confocal microscopy - High-resolution imaging of ZP2 distribution

• Fluorescence-activated cell sorting (FACS) - Isolation of ZP2-expressing cells

For optimal results in immunofluorescence applications, recommended dilutions range from 1:50 to 1:200, though specific optimization may be required based on sample type and preparation method .

How should I design experiments to study ZP2's role in sperm-egg binding using FITC-conjugated antibodies?

For studying ZP2's role in sperm-egg binding, design your experiments with these methodological considerations:

• Sample preparation:

  • For ovulated oocytes: Collect and immediately fix with 4% paraformaldehyde (10 minutes)

  • For tissue sections: Use 5-8 μm paraffin sections of ovarian tissue

• Antibody application:

  • Primary incubation: Apply ZP2 Antibody-FITC at 1:50-1:200 dilution in PBS with 1% BSA

  • Include controls: No-antibody control and isotype control (FITC-conjugated mouse IgG1)

• Sperm-binding assay methodology:

  • Pre-incubate oocytes with antibody (2 hours at room temperature)

  • Introduce capacitated sperm (concentration ~1×10⁶/ml)

  • Assess binding using quantitative fluorescence microscopy

Research has shown that while antibodies to central peptide regions (190-505) of ZP2 bind specifically to the zonae pellucidae, they may not inhibit sperm binding, suggesting functional domains may reside elsewhere .

What controls should be included when using ZP2 Antibody, FITC conjugated for immunolocalization studies?

For rigorous immunolocalization studies using ZP2 Antibody-FITC, include these essential controls:

• Negative controls:

  • Isotype control: FITC-conjugated mouse IgG1 kappa at equivalent concentration

  • Absorption control: Pre-incubate antibody with recombinant ZP2 peptide before staining

  • Secondary-only control: Skip primary antibody step (for protocols using indirect methods)

• Positive controls:

  • Known ZP2-expressing tissues (ovarian sections containing follicles)

  • Cell lines with validated ZP2 expression (such as SH-SY5Y, HL-60, or SW620)

• Specificity controls:

  • Cross-reactivity assessment with other ZP proteins (ZP1, ZP3, ZP4)

  • Comparative staining with alternative ZP2 antibodies recognizing different epitopes

How can I optimize ZP2 Antibody, FITC conjugated for dual immunofluorescence with other zona pellucida proteins?

For dual immunofluorescence involving ZP2-FITC and other zona pellucida proteins, implement this optimized protocol:

• Sequential staining approach:

  • Apply ZP2 Antibody-FITC first (1:100 dilution, 2 hours at room temperature)

  • Wash extensively (5× PBS-Tween washes, 5 minutes each)

  • Apply second primary antibody (anti-ZP1, ZP3, or ZP4) unconjugated or with compatible fluorophore

  • Use appropriate species-specific secondary antibody with distinct emission spectrum (e.g., AlexaFluor 546/594)

• Spectrum optimization:

  • FITC: excitation 495 nm, emission 519 nm (green channel)

  • Partner with red fluorophores (e.g., Cy3, PE, AlexaFluor 546/594)

  • Include single-stained controls to establish proper exposure settings

• Sample preparation:

  • Use 4% paraformaldehyde fixation (10 minutes at room temperature)

  • Permeabilize with 0.2% Triton X-100 (10 minutes)

  • Block with 5% normal serum from secondary antibody species + 1% BSA (1 hour)

This approach minimizes spectral overlap and cross-reactivity between antibodies while maintaining optimal signal quality for each target protein .

What are the recommended protocols for using ZP2 Antibody, FITC conjugated in flow cytometry applications?

For flow cytometry applications with ZP2 Antibody-FITC, follow this optimized protocol:

• Cell preparation:

  • Harvest cells of interest (ovarian tissue cells or cultured cells)

  • Prepare single-cell suspension (enzymatic digestion with collagenase/trypsin)

  • Fix with 2% paraformaldehyde (15 minutes at room temperature)

  • Permeabilize with 0.1% saponin in PBS if targeting intracellular epitopes

• Antibody staining:

  • Block with 2% normal mouse serum, 1% BSA (30 minutes)

  • Incubate with ZP2 Antibody-FITC at 1:100 dilution (45 minutes in dark)

  • Wash 3× with PBS containing 0.1% BSA

• Flow cytometry settings:

  • FITC detection: 488 nm excitation laser, 530/30 nm bandpass filter

  • Compensation: Use single-stained controls if performing multicolor analysis

  • Analysis gates: Set based on isotype control and unstained samples

Expected results: ZP2-positive cells will show increased FITC fluorescence compared to negative controls, with optimal signal at recommended dilutions (1:50-1:200 for flow cytometry) .

How can I troubleshoot weak or non-specific signals when using ZP2 Antibody, FITC conjugated?

For troubleshooting weak or non-specific signals with ZP2 Antibody-FITC, implement this systematic approach:

Compare results with alternative detection methods such as immunohistochemistry with a non-conjugated ZP2 antibody to determine if issues are FITC-specific or target-related .

What pattern of ZP2 localization should be expected in properly conducted immunofluorescence experiments?

In properly conducted immunofluorescence experiments using ZP2 Antibody-FITC, expect the following characteristic patterns:

• Oocyte samples:

  • Intense fluorescence localized to the zona pellucida surrounding the oocyte

  • Uniform, ring-like staining pattern (~10-15 μm thickness in mature oocytes)

  • No staining of oocyte cytoplasm or nucleus

  • Potential partial penetration into zona pellucida rather than just surface staining

• Follicular structures in ovarian sections:

  • Progressive appearance during follicular development

  • Weak signal in primordial/primary follicles

  • Increasing intensity in secondary and antral follicles

  • Strong signal in preovulatory follicles

• Cellular localization in expressing cells:

  • Primarily membrane-associated and secreted protein

  • Possible perinuclear and ER-associated staining in ZP2-producing cells

  • Expected molecular weight on Western blot: 82 kDa (calculated) with observed weight of 68 kDa

Deviations from these patterns may indicate technical issues or interesting biological phenomena warranting further investigation.

How can ZP2 Antibody, FITC conjugated be used to investigate zona pellucida hardening after fertilization?

For investigating zona pellucida hardening post-fertilization using ZP2 Antibody-FITC:

• Experimental design approach:

  • Collect oocytes and embryos at specific time points (unfertilized, 2 hours, 6 hours, 12 hours post-fertilization)

  • Fix with 2% paraformaldehyde to preserve ZP2 cleavage state

  • Stain with ZP2 Antibody-FITC (epitope-specific antibodies may show differential binding based on proteolytic status)

  • Image using confocal microscopy with standardized settings

• Data analysis methodology:

  • Quantify fluorescence intensity changes across time points

  • Measure zona pellucida thickness and structural changes

  • Compare ZP2 staining pattern distribution before and after fertilization

  • Correlate with mechanical resistance measurements (if performed)

Research indicates ZP2 undergoes proteolytic cleavage during fertilization, potentially causing conformational changes that may be detectable as altered binding patterns of domain-specific antibodies. Epitope mapping with different ZP2 antibodies targeting distinct regions can provide insight into structural changes associated with zona hardening .

What methodological approaches can be used to study ZP2 interactions with other zona pellucida proteins using FITC-conjugated antibodies?

To study ZP2 interactions with other zona pellucida proteins using FITC-conjugated antibodies, implement these advanced methodological approaches:

• Proximity ligation assay (PLA) adaptation:

  • Use ZP2 Antibody-FITC as one probe

  • Partner with unconjugated antibodies against ZP1, ZP3, or ZP4

  • Apply appropriate PLA probes to unconjugated antibody

  • Analyze interaction signals in relation to FITC localization

• FRET (Fluorescence Resonance Energy Transfer) analysis:

  • Pair ZP2-FITC (donor) with compatible acceptor fluorophore-conjugated antibodies against other ZP proteins

  • Measure energy transfer efficiency to determine molecular proximity

  • Perform acceptor photobleaching to confirm FRET signals

• Co-immunoprecipitation with fluorescence detection:

  • Use unconjugated ZP2 antibody for pulldown experiments

  • Detect co-precipitated proteins using specific antibodies

  • Verify results with reverse co-IP experiments

  • Correlate with FITC-based localization studies

These approaches provide complementary data on physical associations between ZP2 and other zona pellucida components, offering insights into the molecular architecture of this critical extracellular matrix .

How can researchers use ZP2 Antibody, FITC conjugated to investigate species-specific differences in zona pellucida structure?

For comparative studies of species-specific zona pellucida structures using ZP2 Antibody-FITC:

• Cross-species reactivity assessment:

  • Test antibody reactivity across mammalian species (human, mouse, rat, bovine, porcine)

  • Determine optimal working dilutions for each species (starting with 1:100)

  • Document cross-reactivity patterns based on epitope conservation

• Comparative analytical methodology:

  • Standardize imaging parameters across species samples

  • Quantify:

    • ZP2 distribution patterns (uniform vs. layered)

    • Fluorescence intensity profiles across zona thickness

    • Co-localization coefficients with other ZP proteins

• Structural analysis correlation:

  • Compare antibody binding patterns with ultrastructural features (from EM studies)

  • Analyze glycosylation differences using lectin co-staining

  • Correlate structural variations with functional differences in fertilization

Current research suggests ZP2 Antibody (C-7) primarily detects human ZP2, while other antibodies like the Rabbit Polyclonal (CAB10126) show broader reactivity with human, mouse, and rat samples. These reactivity differences can be leveraged to explore evolutionary conservation and specialization of zona pellucida architecture across species .