ZMPSTE24 Antibody, FITC conjugated

What is ZMPSTE24 and what are its biological functions?

ZMPSTE24 (Zinc metalloproteinase STE24) is a transmembrane metalloprotease with multiple critical cellular functions. It serves as a proteolytic enzyme that removes the C-terminal three residues of farnesylated proteins. Its primary functions include:

• Processing lamin A/LMNA on the inner nuclear membrane, which is essential for nuclear envelope integrity and function .

• Clearing clogged translocons on the endoplasmic reticulum to maintain proper protein trafficking .

• Acting as a broad-spectrum antiviral protein that restricts enveloped RNA and DNA viruses, including influenza A, Zika, Ebola, Sindbis, vesicular stomatitis, cowpox, and vaccinia viruses .

• Functioning as an effector in the interferon-induced transmembrane protein (IFITM) pathway to hinder viruses from breaching the endosomal barrier by modulating membrane fluidity .

Interestingly, while ZMPSTE24 is a metalloprotease, its antiviral functions operate independently of its protease activity, suggesting distinct functional domains within the protein .

What are the key specifications of commercially available ZMPSTE24 Antibody, FITC conjugated?

Various FITC-conjugated ZMPSTE24 antibodies are available with distinct specifications that determine their research applications:

When selecting an antibody, researchers should consider the target species, application requirements, and the specific region of ZMPSTE24 relevant to their research question .

How should ZMPSTE24 Antibody, FITC conjugated be stored to maintain optimal activity?

Proper storage of FITC-conjugated antibodies is crucial for maintaining their fluorescent properties and binding specificity over time:

• Short-term storage (up to 2 weeks): Maintain refrigerated at 2-8°C in the dark to prevent photobleaching of the FITC conjugate .

• Long-term storage: Store at -20°C in small aliquots to prevent freeze-thaw cycles that can damage both antibody function and fluorescent properties .

• Buffer conditions: The antibody is typically supplied in stabilization buffer, sometimes containing preservatives such as 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4 .

• Light protection: FITC is photosensitive, so always store and handle these antibodies protected from light exposure. Amber tubes or aluminum foil wrapping is recommended for all storage containers .

• Avoid repeated freeze-thaw cycles: Create multiple single-use aliquots before freezing to maintain antibody integrity and fluorescence intensity over time .

For maximum shelf life, follow the manufacturer's specific recommendations, as formulations may vary between suppliers.

What experimental controls should be included when working with ZMPSTE24 Antibody, FITC conjugated?

Implementing appropriate controls is essential for generating reliable and interpretable data with FITC-conjugated ZMPSTE24 antibodies:

• Negative controls:

  • Isotype control: Use FITC-conjugated rabbit IgG (non-specific) at the same concentration as your ZMPSTE24 antibody to assess non-specific binding .

  • Secondary antibody-only control (if using indirect detection methods).

  • Cells known to not express ZMPSTE24 or ZMPSTE24 knockout cells.

• Positive controls:

  • Cell lines with confirmed ZMPSTE24 expression (based on literature).

  • Recombinant ZMPSTE24 protein for western blot validation.

  • IFN-β-stimulated cells for experiments investigating viral restriction functions (note that unlike IFITM proteins, ZMPSTE24 expression is not upregulated by IFN-β stimulation) .

• Fluorescence controls:

  • Autofluorescence control: Unstained samples to determine baseline fluorescence.

  • Single-color controls for compensation when performing multicolor flow cytometry.

  • FITC quenching control to account for photobleaching during extended imaging sessions.

• Specificity validation:

  • Antibody blocking with immunizing peptide (if available).

  • ZMPSTE24 knockdown/knockout validation if suitable genetic systems are available .

Implementing these controls will help distinguish specific from non-specific signals and ensure accurate interpretation of experimental results.

How can ZMPSTE24 Antibody, FITC conjugated be optimized for different applications?

Optimization strategies vary by application method, requiring different approaches for maximum sensitivity and specificity:

For Western Blotting (1:500 dilution recommended) :

• Protein extraction optimization: Use detergent-based buffers (e.g., RIPA) with protease inhibitors to efficiently extract this transmembrane protein.

• Sample preparation: Heat samples at 70°C instead of boiling to prevent aggregation of this membrane protein.

• Loading control selection: Use membrane protein markers rather than cytosolic proteins for normalization.

• Detection optimization: For weak signals, consider using enhanced chemiluminescence substrate compatible with fluorescence detection.

• Exposure time adjustment: FITC signal may require longer exposure times than other fluorophores, balancing sensitivity against background.

For ELISA (1:10,000 dilution recommended) :

• Coating buffer optimization: Test different coating buffers (carbonate/bicarbonate pH 9.6 vs. PBS pH 7.4) for optimal antigen presentation.

• Blocking agent selection: Compare BSA vs. non-fat milk to minimize background while maintaining specific signal.

• Incubation temperature: Compare room temperature vs. 4°C incubation for optimal signal-to-noise ratio.

• Signal enhancement: Consider avidin-biotin amplification if direct FITC detection provides insufficient sensitivity.

• Standard curve generation: Use recombinant ZMPSTE24 protein at known concentrations for quantification.

For Immunofluorescence:

• Fixation method optimization: Compare paraformaldehyde vs. methanol fixation for preserving ZMPSTE24 epitopes.

• Permeabilization refinement: Test different detergents (Triton X-100, saponin) at various concentrations.

• Antibody concentration titration: Perform serial dilutions to identify optimal signal-to-noise ratio.

• Signal amplification: Consider tyramide signal amplification for detecting low-abundance ZMPSTE24.

• Mounting media selection: Use anti-fade mounting media specifically designed for FITC to prevent photobleaching during analysis .

What techniques can be used to study ZMPSTE24-IFITM protein interactions using FITC-conjugated antibodies?

Recent research has identified ZMPSTE24 as an important interacting partner with IFITM proteins in antiviral responses. Several techniques can leverage FITC-conjugated ZMPSTE24 antibodies to investigate these interactions:

• Co-immunoprecipitation coupled with fluorescence detection:

  • Precipitate IFITM proteins using specific antibodies and detect co-precipitated ZMPSTE24 using FITC-conjugated anti-ZMPSTE24.

  • Quantify fluorescence intensity directly from the immunoprecipitated complex to measure interaction strength.

  • Research has confirmed that ZMPSTE24 specifically binds IFITM1, IFITM2, and IFITM3 through co-IP studies .

• Fluorescence Resonance Energy Transfer (FRET):

  • Label IFITM proteins with a FRET-compatible fluorophore (e.g., Cy3, rhodamine) that can accept energy from FITC.

  • Measure energy transfer efficiency to determine proximity between ZMPSTE24 and IFITM proteins.

  • Calculate FRET efficiency using spectral imaging to quantify interaction dynamics.

• Proximity Ligation Assay (PLA):

  • Combine FITC-conjugated anti-ZMPSTE24 with non-fluorescent anti-IFITM antibodies.

  • Use complementary oligonucleotide-labeled secondary antibodies.

  • Amplify signal only when proteins are in close proximity (<40 nm), providing spatial resolution of interactions.

• Fluorescence Recovery After Photobleaching (FRAP):

  • Photobleach FITC-labeled ZMPSTE24 in specific cellular compartments.

  • Measure recovery rate in wild-type vs. IFITM-knockout cells to assess how IFITM proteins influence ZMPSTE24 mobility.

  • Analyze diffusion coefficients to understand interaction dynamics.

• Live cell imaging:

  • Complement FITC-ZMPSTE24 antibody (for fixed cells) with fluorescent protein-tagged ZMPSTE24 (for live cells).

  • Correlate antibody staining patterns with live cell dynamics.

  • Track ZMPSTE24-IFITM interaction during viral challenge using time-lapse microscopy .

How can researchers investigate the antiviral functions of ZMPSTE24 using fluorescent antibodies?

ZMPSTE24 exhibits broad-spectrum antiviral activity against multiple enveloped viruses. FITC-conjugated ZMPSTE24 antibodies can be employed in various experimental approaches to elucidate these functions:

• Viral challenge assays with fluorescence readout:

  • Infect cells with reporter viruses expressing fluorescent proteins.

  • Simultaneously detect ZMPSTE24 localization using FITC-conjugated antibodies.

  • Quantify correlation between ZMPSTE24 expression levels and viral restriction.

  • Studies have shown ZMPSTE24 restricts influenza A, Zika, Ebola, Sindbis, vesicular stomatitis, cowpox, and vaccinia viruses, but not murine leukemia or adenovirus .

• High-content imaging of virus-cell interactions:

  • Use FITC-ZMPSTE24 antibodies alongside viral protein staining.

  • Perform automated image analysis to quantify colocalization during viral entry.

  • Measure spatial relationships between ZMPSTE24, viral particles, and cellular compartments.

  • Track changes in ZMPSTE24 distribution during different stages of infection.

• Structure-function analysis using mutant ZMPSTE24:

  • Generate cells expressing catalytically inactive ZMPSTE24 mutants.

  • Compare antiviral activity between wild-type and mutant ZMPSTE24 using FITC-antibody detection.

  • Research has demonstrated that ZMPSTE24's protease activity is dispensable for its antiviral function .

• Membrane fluidity measurements:

  • Use FITC-ZMPSTE24 antibodies alongside membrane fluidity probes.

  • Correlate ZMPSTE24 expression with changes in membrane properties.

  • ZMPSTE24 controls the IFITM antiviral pathway by modulating membrane fluidity to hinder viruses from breaching the endosomal barrier .

• IFITM-dependent restriction analysis:

  • Compare viral restriction in IFITM-expressing versus IFITM-knockout cells.

  • Use FITC-ZMPSTE24 antibodies to track ZMPSTE24 recruitment to viral entry sites.

  • Research indicates ZMPSTE24 expression is necessary for IFITM antiviral activity .

What are the considerations for using ZMPSTE24 Antibody, FITC conjugated in multicolor imaging studies?

Multicolor imaging with FITC-conjugated ZMPSTE24 antibodies requires careful experimental design to achieve clear signal separation and meaningful biological insights:

• Fluorophore selection and spectral overlap:

  • FITC excitation maximum: ~490 nm, emission maximum: ~525 nm

  • Avoid fluorophores with significant spectral overlap, such as GFP or BODIPY-FL

  • Recommended compatible fluorophores: DAPI (nuclei), Cy3/TRITC (separate target), Cy5/Alexa647 (separate target)

• Compensation and linear unmixing:

  • Collect single-color controls for each fluorophore

  • Perform spectral unmixing using imaging software to separate overlapping signals

  • Consider acquiring lambda stacks on confocal microscopes for precise signal separation

• Sequential acquisition strategies:

  • Image FITC channel first to minimize photobleaching effects

  • Use sequential rather than simultaneous acquisition when possible

  • Employ narrow bandpass filters to minimize bleed-through

• Optimal counterstains for subcellular localization:

  Subcellular Compartment	Recommended Counterstain	Rationale for ZMPSTE24 Studies
  Nuclear envelope	Lamin B (Far-red channel)	ZMPSTE24 processes lamin A at inner nuclear membrane
  Endoplasmic reticulum	Calnexin (Cy3/TRITC)	ZMPSTE24 clears clogged translocons on ER
  Endosomes	Rab5/Rab7 (Cy3/TRITC)	ZMPSTE24 functions in endosomal viral restriction
  IFITM proteins	IFITM1/2/3 (Far-red channel)	ZMPSTE24 interacts with IFITM proteins
  Viral proteins	Virus-specific markers (Far-red)	Visualize ZMPSTE24-virus interactions

• Challenges and solutions:

  • Autofluorescence: Use spectral imaging and linear unmixing to separate specific signal

  • Photobleaching: Minimize exposure times and use antifade mounting media specifically compatible with FITC

  • Signal-to-noise: Consider signal amplification methods if ZMPSTE24 expression is low

What are common issues encountered when using ZMPSTE24 Antibody, FITC conjugated and how can they be resolved?

Researchers often encounter several challenges when working with FITC-conjugated ZMPSTE24 antibodies. Here are systematic approaches to address these issues:

• Low signal intensity:

  • Problem: Insufficient detection of ZMPSTE24 despite proper technique

  • Causes: Low abundance protein, epitope masking, antibody degradation

  • Solutions:

    • Increase antibody concentration (try 2-5× recommended concentration)

    • Extend incubation time (overnight at 4°C)

    • Try alternative fixation methods that better preserve epitope accessibility

    • Implement signal amplification (tyramide signal amplification, avidin-biotin systems)

    • Ensure FITC fluorescence is preserved by using appropriate antifade reagents

• High background fluorescence:

  • Problem: Poor signal-to-noise ratio making specific signal difficult to distinguish

  • Causes: Non-specific binding, insufficient blocking, autofluorescence

  • Solutions:

    • Optimize blocking (test 5% BSA vs. 5% normal serum from same species as secondary antibody)

    • Include 0.1-0.3% Triton X-100 in antibody diluent to reduce non-specific binding

    • Use longer/more stringent washing steps (5× 5 min washes)

    • For tissues, include autofluorescence quenching step (0.1% Sudan Black B in 70% ethanol)

    • Reduce antibody concentration and extend incubation time

• Inconsistent staining patterns:

  • Problem: Variable or contradictory localization patterns

  • Causes: Antibody cross-reactivity, fixation artifacts, sample preparation issues

  • Solutions:

    • Validate specificity using ZMPSTE24 knockout/knockdown controls

    • Standardize fixation and permeabilization protocols

    • Compare results with non-FITC ZMPSTE24 antibodies recognizing different epitopes

    • Use multiple antibodies targeting different regions of ZMPSTE24 (such as AA 217-347 vs. AA 450-480)

• Poor reproducibility in flow cytometry:

  • Problem: Variable detection of ZMPSTE24-positive populations

  • Causes: Inconsistent permeabilization, suboptimal antibody concentration

  • Solutions:

    • Standardize permeabilization (compare 0.1% saponin vs. 0.1% Triton X-100)

    • Titrate antibody carefully (create dilution series from 1:100 to 1:2000)

    • Include viability dye to exclude dead cells that may bind antibodies non-specifically

    • Use freshly prepared samples when possible

How can researchers differentiate between protease-dependent and protease-independent functions of ZMPSTE24?

ZMPSTE24 exhibits dual functionality—proteolytic processing of proteins like lamin A and protease-independent antiviral activity. Distinguishing between these functions requires specialized experimental approaches:

• Genetic engineering approaches:

  • Generate catalytically inactive ZMPSTE24 mutants (e.g., mutations in the HEXXH metalloprotease motif)

  • Express these mutants in ZMPSTE24-knockout cells

  • Use FITC-conjugated anti-ZMPSTE24 to confirm expression and localization

  • Assess both proteolytic functions (lamin A processing) and antiviral activities

  • Research has confirmed that ZMPSTE24's antiviral function is independent of its protease activity

• Pharmacological inhibition:

  • Apply metalloprotease inhibitors (e.g., 1,10-phenanthroline) at concentrations that specifically inhibit ZMPSTE24

  • Monitor both proteolytic processing and antiviral functions

  • Use FITC-conjugated ZMPSTE24 antibodies to confirm protein expression remains unchanged

  • Compare results with genetic knockout/mutation approaches

• Imaging-based differentiation:

  • Perform dual immunofluorescence with FITC-ZMPSTE24 antibody and antibodies against:

    • Prelamin A (substrate accumulates when proteolytic function is inhibited)

    • Viral proteins (to assess antiviral function)

  • Quantify colocalization under different conditions (wild-type vs. mutant ZMPSTE24)

  • Track dynamic changes during viral infection using time-lapse imaging

• Domain-specific functional mapping:

  • Create domain deletion mutants of ZMPSTE24

  • Use FITC-conjugated antibodies to confirm expression and localization

  • Map which domains are required for proteolytic vs. antiviral functions

  • Consider the possibility that antiviral function involves ZMPSTE24 interaction with IFITM proteins

• Interaction partner analysis:

  • Perform co-immunoprecipitation of wild-type vs. catalytically inactive ZMPSTE24

  • Use FITC-conjugated ZMPSTE24 antibodies to confirm precipitation

  • Identify differential binding partners using mass spectrometry

  • Focus particularly on IFITM protein interactions, which have been shown to be important for ZMPSTE24's antiviral function

What considerations are important when using ZMPSTE24 Antibody, FITC conjugated in in vivo studies?

In vivo applications of FITC-conjugated ZMPSTE24 antibodies present unique challenges that require specialized approaches:

• Antibody delivery considerations:

  • Blood-brain barrier penetration may be limited for antibodies

  • Consider intracerebroventricular injection for CNS studies

  • For peripheral tissues, optimize injection volume and timing

  • FITC's relatively short wavelength may limit tissue penetration for in vivo imaging

• In vivo infection models:

  • ZMPSTE24-deficient mice display higher viral burdens, enhanced cytokine production, and increased mortality after influenza infection

  • When using FITC-conjugated antibodies for ex vivo analysis:

    • Collect tissues at appropriate timepoints post-infection

    • Process rapidly to preserve FITC fluorescence

    • Include autofluorescence controls (especially for lung tissue)

• Optimal tissue processing for ex vivo analysis:

  • Fix tissues in 4% paraformaldehyde for minimal autofluorescence

  • Consider shorter fixation times (4-8 hours) to preserve FITC signal

  • Use sucrose cryoprotection before frozen sectioning

  • For paraffin sections, be aware that the deparaffinization process may reduce FITC signal

  • Perform antigen retrieval carefully to avoid further fluorescence loss

• Quantification approaches:

  • Use automated whole-slide scanning for unbiased analysis

  • Implement spectral unmixing to separate FITC signal from tissue autofluorescence

  • Consider counterstaining with far-red fluorophores to avoid spectral overlap

  • Include appropriate controls in each experimental batch:

    • ZMPSTE24-knockout tissues as negative controls

    • Isotype-FITC antibodies to assess non-specific binding

• Ethical and practical considerations:

  • Calculate minimum animal numbers needed for statistical significance

  • Consider ex vivo validation before proceeding to in vivo studies

  • Recognize that ZMPSTE24 deficiency in mice models human progeroid syndromes, which may confound interpretation of some experimental results

How can ZMPSTE24 Antibody, FITC conjugated be utilized to investigate membrane dynamics during viral infection?

Recent research indicates ZMPSTE24 plays a role in modulating membrane fluidity during viral infections. FITC-conjugated antibodies offer unique opportunities to explore these dynamics:

• Membrane microdomain analysis:

  • Use FITC-ZMPSTE24 antibodies alongside lipid raft markers (CTxB-647, caveolin-1)

  • Track redistribution following viral challenge

  • Perform quantitative colocalization analysis to determine enrichment in specific membrane domains

  • Apply super-resolution microscopy (STED, STORM) to resolve nanoscale organization

  • ZMPSTE24 has been shown to modulate membrane fluidity to prevent viruses from breaching the endosomal barrier

• Live-cell compatible approaches:

  • Combine fixed-cell FITC-antibody imaging with live-cell studies using:

    • Fluorescent protein-tagged ZMPSTE24 constructs

    • Membrane fluidity probes (Laurdan, di-4-ANEPPDHQ)

  • Correlate ZMPSTE24 distribution with biophysical membrane properties

  • Implement FRAP (Fluorescence Recovery After Photobleaching) to measure lateral mobility

• High-resolution spatiotemporal analysis:

  • Track endosomal maturation during viral entry using FITC-ZMPSTE24 with Rab5/Rab7/LAMP1 markers

  • Perform time-course fixation to capture key stages of viral entry

  • Quantify ZMPSTE24 redistribution relative to viral particles and endosomal markers

  • Use 3D reconstruction to visualize complete spatial relationships

• IFITM-dependent membrane remodeling:

  • Compare membrane organization in wild-type vs. IFITM-knockout cells

  • Assess ZMPSTE24 distribution using FITC-conjugated antibodies

  • Correlate with viral restriction phenotypes

  • Research has established that ZMPSTE24 expression is necessary for IFITM antiviral activity

• Viral fusion inhibition mechanisms:

  • Apply fluorescently labeled virus particles to cells

  • Use FITC-ZMPSTE24 antibodies in fixed-timepoint studies

  • Determine if ZMPSTE24 directly blocks hemifusion, pore formation, or content mixing

  • Correlate with membrane rigidity measurements to confirm mechanistic models

What specialized techniques can be used to study ZMPSTE24 in rare cell populations or tissues?

Investigating ZMPSTE24 in rare cell populations or specific tissue microenvironments requires specialized approaches that maximize sensitivity and specificity:

• Flow cytometry and cell sorting:

  • Use FITC-ZMPSTE24 antibodies in combination with lineage markers

  • Implement compensation controls to account for spectral overlap

  • Sort ZMPSTE24-high versus ZMPSTE24-low populations for downstream analysis

  • Consider using branched DNA signal amplification for low-abundance detection

  • Recommended dilution for flow cytometry: 1:500-1:1000

• Mass cytometry (CyTOF) integration:

  • Combine FITC-ZMPSTE24 detection with metal-tagged antibodies

  • Develop a panel of 30+ markers to comprehensively phenotype cells

  • Implement dimensionality reduction algorithms (tSNE, UMAP) for visualization

  • Create cellular behavioral maps to correlate ZMPSTE24 expression with functional states

• Laser capture microdissection with immunofluorescence:

  • Perform FITC-ZMPSTE24 immunostaining on frozen tissue sections

  • Identify regions/cells of interest based on ZMPSTE24 expression

  • Microdissect specific populations for downstream molecular analysis

  • Protect FITC from photobleaching during microscopy by minimizing exposure

• High-parameter imaging approaches:

  • Implement imaging mass cytometry or Codex for 40+ parameter imaging

  • Include FITC-ZMPSTE24 antibody in the panel

  • Create spatial maps of ZMPSTE24 expression in tissue microenvironments

  • Correlate with disease states, viral tropism, or developmental stages

• Single-cell proteogenomic integration:

  • Sort cells based on FITC-ZMPSTE24 levels

  • Perform single-cell RNA-seq on sorted populations

  • Correlate protein expression (from antibody) with transcript levels

  • Identify regulatory networks associated with ZMPSTE24 expression

  • This approach is particularly valuable for understanding heterogeneous responses to viral infection