INTS13 Antibody, FITC conjugated

What is INTS13 and why is it important in cellular research?

INTS13 (Integrator Complex Subunit 13) functions as a crucial regulator of the mitotic cell cycle and development. At prophase, it is required for dynein anchoring to the nuclear envelope, which is important for proper centrosome-nucleus coupling. During G2/M phase, INTS13 may be essential for proper spindle formation and cytokinesis execution . The fluorescein isothiocyanate (FITC) conjugation allows visualization of this protein in various experimental contexts, particularly in fluorescence microscopy and flow cytometry applications. When studying cell cycle regulation, INTS13 antibodies provide valuable insights into nuclear dynamics and mitotic progression.

What applications are most suitable for FITC-conjugated INTS13 antibodies?

FITC-conjugated INTS13 antibodies are primarily utilized in flow cytometry, immunohistochemistry, and immunofluorescence microscopy applications. The FITC fluorophore has an excitation maximum at approximately 495 nm and emission maximum around 519 nm, making it compatible with standard fluorescence detection systems . These antibodies are particularly valuable for:

• Tracking INTS13 expression during cell cycle progression

• Colocalization studies with other nuclear proteins

• Quantitative analysis of INTS13 expression levels in different cell populations

• Immunophenotyping of cell types based on INTS13 expression patterns

The optimal dilution for these applications should be experimentally determined for each specific research context, as sensitivity requirements may vary across different experimental designs.

What are the recommended storage conditions for maintaining FITC-conjugated INTS13 antibody activity?

To preserve the fluorescence intensity and binding activity of FITC-conjugated INTS13 antibodies, proper storage conditions are essential. Store the antibody at 4°C in the dark for short-term storage (1-2 weeks) or at -20°C to -80°C for long-term storage . Avoid repeated freeze-thaw cycles as they can lead to protein denaturation and fluorophore degradation. When stored at -20°C or -80°C, aliquoting the antibody into smaller volumes before freezing is recommended to minimize freeze-thaw cycles. Additionally, FITC is sensitive to photobleaching, so minimize exposure to light during both storage and experimental procedures. Some formulations contain preservatives such as sodium azide (0.05-0.1%) or ProClin (0.03%) to prevent microbial contamination .

How does FITC conjugation affect the binding properties of INTS13 antibodies?

FITC conjugation introduces fluorescent molecules to the antibody structure, which can potentially impact antigen recognition and binding kinetics. The conjugation process typically targets primary amine groups (lysine residues) on the antibody. While manufacturers optimize conjugation protocols to minimize interference with antigen binding sites, researchers should be aware of several considerations:

• Binding affinity: FITC conjugation may slightly reduce antibody affinity compared to unconjugated versions

• Background fluorescence: Over-conjugation can increase non-specific binding and background signal

• Steric hindrance: The FITC molecule may cause steric hindrance when the epitope is in a spatially restricted environment

Validation experiments comparing unconjugated and FITC-conjugated INTS13 antibodies in your specific experimental system are recommended to assess any potential differences in binding properties. When possible, include appropriate isotype controls with matching FITC conjugation to distinguish between specific and non-specific binding patterns.

How can I optimize FITC-conjugated INTS13 antibody staining for confocal microscopy to study nuclear localization patterns?

Optimizing FITC-conjugated INTS13 antibody staining for confocal microscopy requires careful attention to fixation methods, permeabilization protocols, and imaging parameters. The following methodological approach is recommended:

• Fixation optimization: Compare paraformaldehyde (2-4%) with methanol fixation to determine which better preserves INTS13 epitopes while maintaining nuclear architecture.

• Permeabilization testing: Nuclear proteins often require optimized permeabilization. Test a gradient of detergent concentrations (0.1-0.5% Triton X-100 or 0.05-0.2% saponin) to identify optimal conditions.

• Blocking protocol: Use a dual blocking approach with 5-10% normal serum and 1-3% BSA to minimize background fluorescence.

• Antibody titration: Perform a dilution series (typically 1:50 to 1:500) of FITC-conjugated INTS13 antibody to determine optimal signal-to-noise ratio.

• Counter-staining strategy: Combine DAPI nuclear staining with additional markers for nuclear substructures relevant to INTS13 function.

• Imaging parameters:

  • Use 488 nm laser excitation for FITC visualization

  • Adjust pinhole size to 1 Airy unit for optimal confocal sectioning

  • Employ sequential scanning when using multiple fluorophores to prevent bleed-through

  • Capture z-stacks at 0.3-0.5 μm intervals for 3D reconstruction of nuclear distribution

• Controls:

  • Include FITC-conjugated isotype control antibodies

  • Perform peptide competition assays to confirm signal specificity

  • Include cells known to be negative for INTS13 expression

This comprehensive approach allows for high-resolution analysis of INTS13 nuclear distribution patterns while minimizing artifacts and non-specific signals.

What strategies can resolve contradictory data between FITC-conjugated INTS13 antibody signals and RNA expression levels?

Discrepancies between protein detection using FITC-conjugated INTS13 antibodies and corresponding mRNA expression levels represent a common challenge in molecular biology research. A systematic approach to resolve such contradictions includes:

• Verify antibody specificity:

  • Perform western blot analysis using the same antibody (unconjugated version)

  • Conduct siRNA/shRNA knockdown experiments to confirm signal reduction

  • Use alternative antibodies targeting different INTS13 epitopes

  • Employ IP-MS (immunoprecipitation followed by mass spectrometry) to confirm antibody specificity

• Assess post-transcriptional regulation:

  • Measure INTS13 protein half-life using cycloheximide chase experiments

  • Investigate microRNA-mediated regulation of INTS13 translation

  • Examine alternative splicing patterns that might affect epitope availability

• Evaluate technical variables:

  • Optimize fixation and permeabilization conditions for different cell types

  • Test different RNA isolation methods to ensure complete extraction

  • Verify primer specificity for qPCR analysis

  • Consider single-cell analysis to address population heterogeneity

• Quantitative comparison:

  Analysis Method	Advantages	Limitations	Correlation with Other Methods
  Flow cytometry with FITC-INTS13	Single-cell resolution, quantitative	Loses spatial information	Moderate correlation with western blot
  IF microscopy with FITC-INTS13	Preserves spatial information	Semi-quantitative	Variable correlation with RNA-seq
  RT-qPCR for INTS13 mRNA	High sensitivity	No protein information	Often poor correlation with protein levels
  RNA-seq	Comprehensive transcriptome	Bulk measurement	Moderate correlation with proteomics
  Western blot	Protein size confirmation	Loss of spatial information	Variable correlation with IF microscopy

• Biological interpretation:

  • Consider cell cycle-dependent regulation of INTS13 (both transcriptional and post-translational)

  • Evaluate subcellular localization changes that might affect antibody accessibility

  • Investigate protein-protein interactions that could mask antibody epitopes

By systematically addressing these factors, researchers can reconcile contradictory data and develop a more nuanced understanding of INTS13 biology.

How can I design multiplexed flow cytometry panels incorporating FITC-conjugated INTS13 antibody for cell cycle analysis?

Designing effective multiplexed flow cytometry panels that include FITC-conjugated INTS13 antibody for cell cycle analysis requires careful consideration of fluorophore compatibility, compensation requirements, and biological relevance. The following methodological approach is recommended:

• Panel design considerations:

  • FITC (excitation: 495 nm, emission: 519 nm) occupies the green fluorescence channel (FL1 on most cytometers)

  • Avoid fluorophores with significant spectral overlap with FITC (e.g., PE, Alexa Fluor 488)

  • Compatible fluorophores include APC, Pacific Blue, PE-Cy5, and PE-Cy7

  • Reserve brightest fluorophores (PE, APC) for markers with lowest expression levels

• Proposed panel for cell cycle analysis with INTS13:

  Target	Fluorophore	Excitation (nm)	Emission (nm)	Purpose
  INTS13	FITC	495	519	Target protein analysis
  DNA content	PI or 7-AAD	536	617	Cell cycle phase identification
  pH3 (Ser10)	Pacific Blue	401	452	Mitotic cell identification
  Ki-67	APC	650	660	Proliferating cell identification
  Cleaved PARP	PE-Cy7	496/743	578/767	Apoptotic cell exclusion

• Sample preparation protocol:

  • Fix cells with 2-4% paraformaldehyde (10 minutes, room temperature)

  • Permeabilize with 0.1% Triton X-100 in PBS (5-10 minutes, room temperature)

  • Block with 3% BSA in PBS (30 minutes, room temperature)

  • Stain with optimized concentrations of antibody cocktail (60 minutes, 4°C, protected from light)

  • Wash twice with PBS containing 1% BSA

  • Resuspend in appropriate DNA staining solution (if using PI or 7-AAD)

  • Analyze within 4 hours or fix additionally with 1% paraformaldehyde for later analysis

• Instrument setup and compensation:

  • Run single-stained controls for each fluorophore

  • Include FMO (Fluorescence Minus One) controls to identify gating boundaries

  • Apply appropriate compensation matrix to correct for spectral overlap

  • Establish voltage settings that position negative populations appropriately

• Analysis strategy:

  • Gate on single cells using FSC-A vs. FSC-H

  • Exclude dead cells and debris using FSC/SSC properties

  • Gate on cell cycle phases using DNA content histogram

  • Analyze INTS13-FITC expression within each cell cycle phase

  • Compare INTS13 expression patterns with proliferation markers (Ki-67) and mitotic markers (pH3)

This comprehensive approach enables detailed characterization of INTS13 expression dynamics throughout the cell cycle while controlling for technical variables and biological heterogeneity.

Why might FITC-conjugated INTS13 antibody show weak or no signal in flow cytometry despite successful staining with other antibodies?

Weak or absent signals from FITC-conjugated INTS13 antibodies in flow cytometry experiments can result from multiple technical and biological factors. A systematic troubleshooting approach includes:

• Antibody-specific issues:

  • Verify antibody viability: FITC is sensitive to photobleaching and pH changes. Ensure proper storage in the dark at 4°C and avoid repeated freeze-thaw cycles

  • Check conjugation quality: Over-conjugation or sub-optimal conjugation can affect binding efficiency and fluorescence intensity

  • Confirm epitope accessibility: The targeted region (e.g., AA 573-706 in INTS13) may be masked in certain fixation conditions

  • Validate antibody with positive control samples known to express INTS13

• Protocol optimization:

  • Adjust antibody concentration: Titrate the antibody using 2-fold serial dilutions to find optimal concentration

  • Modify fixation/permeabilization: Test different fixatives (PFA vs. methanol) and permeabilization agents (saponin vs. Triton X-100)

  • Extend incubation time: Increase staining time to 1-2 hours at 4°C

  • Include protein transport inhibitors (like Brefeldin A) if working with stimulated cells

• Instrument and technical considerations:

  • Check cytometer settings: Verify that the 488 nm laser is functioning properly and detector voltages are appropriate

  • Assess for compensation issues: FITC signal may be unnecessarily compensated away if spillover matrices are incorrect

  • Evaluate for quenching: Certain buffers or other staining components may quench FITC fluorescence

• Biological factors:

  • Consider cell cycle dependence: INTS13 expression may vary throughout the cell cycle, potentially resulting in subpopulations with different signal intensities

  • Assess protein turnover: INTS13 may undergo rapid degradation under certain experimental conditions

  • Evaluate subcellular localization: Nuclear proteins like INTS13 may require specialized permeabilization protocols for optimal detection

If these approaches do not resolve the issue, consider alternative detection strategies such as indirect staining with unconjugated primary antibody followed by FITC-conjugated secondary antibody to amplify the signal.

How can I minimize autofluorescence interference when using FITC-conjugated INTS13 antibody in tissues with high natural green fluorescence?

Autofluorescence presents a significant challenge when using FITC-conjugated antibodies in tissues with intrinsic green fluorescence, such as those containing lipofuscin, elastin, collagen, or NADPH. The following comprehensive strategy can help minimize autofluorescence interference:

• Pre-treatment methods:

  • Apply Sudan Black B (0.1-0.3% in 70% ethanol) for 10-20 minutes after antibody staining to quench lipofuscin autofluorescence

  • Treat with sodium borohydride (0.1-1% in PBS) for 5-10 minutes before blocking to reduce aldehyde-induced fluorescence

  • Use CuSO₄ (1-5 mM in 50 mM ammonium acetate buffer) to quench extracellular matrix autofluorescence

  • Apply photobleaching by exposing the sample to the excitation light source for 10-15 minutes before antibody staining

• Alternative fluorophore strategies:

  • Consider antibody re-conjugation with fluorophores in different spectral ranges (e.g., APC, PE-Cy7)

  • Use fluorophores with large Stokes shifts to better distinguish from autofluorescence

  • Employ quantum dots as alternative labels that offer narrow emission spectra and resistance to photobleaching

• Microscopy and imaging optimization:

  • Utilize spectral unmixing algorithms available on confocal microscopy systems

  • Implement time-gated detection to exploit the typically shorter fluorescence lifetime of FITC compared to autofluorescence

  • Apply narrow bandpass filters centered precisely on FITC emission maximum

  • Use linear unmixing based on reference spectra of autofluorescence and FITC signals

• Advanced signal processing:

  Approach	Methodology	Advantages	Limitations
  Spectral unmixing	Acquisition of spectral signatures followed by computational separation	Preserves signal integrity	Requires specialized equipment
  Autofluorescence subtraction	Imaging of unstained sample to create subtraction mask	Simple implementation	May create artifacts
  Lifetime imaging (FLIM)	Measurement of fluorescence decay times	Excellent separation capability	Requires specialized instrumentation
  Multi-excitation comparison	Imaging with different excitation wavelengths	Distinguishes based on excitation properties	Requires multiple laser lines

• Flow cytometry considerations:

  • Include unstained controls to establish autofluorescence baseline

  • Use 530/30 nm bandpass filter for optimal FITC detection with minimal autofluorescence overlap

  • Consider fluorescence-minus-one (FMO) controls with all antibodies except FITC-INTS13

  • Apply compensation algorithms specifically designed for autofluorescence correction

By implementing these strategies, researchers can effectively distinguish specific FITC-INTS13 antibody signals from tissue autofluorescence, enhancing the reliability and sensitivity of their analyses.

What are the best approaches for validating FITC-conjugated INTS13 antibody specificity in novel cell types or experimental models?

Validating antibody specificity is a critical step when introducing FITC-conjugated INTS13 antibodies to novel experimental systems. A comprehensive validation strategy should include multiple complementary approaches:

• Genetic validation methods:

  • CRISPR/Cas9 knockout: Generate INTS13 knockout cell lines and confirm loss of antibody staining

  • siRNA/shRNA knockdown: Demonstrate proportional reduction in antibody signal with decreased INTS13 expression

  • Overexpression systems: Show increased signal intensity in cells transfected with INTS13 expression vectors

  • Rescue experiments: Restore antibody staining by expressing INTS13 in knockout models

• Biochemical validation approaches:

  • Western blot correlation: Confirm that flow cytometry or microscopy signal intensity correlates with band intensity on western blots using the same (unconjugated) antibody

  • Immunoprecipitation: Verify that the antibody can specifically pull down INTS13 protein

  • Mass spectrometry: Confirm the identity of proteins immunoprecipitated by the antibody

  • Peptide competition: Demonstrate signal reduction when antibody is pre-incubated with the immunizing peptide

• Comparative antibody analysis:

  • Multiple antibody concordance: Test different antibodies targeting distinct INTS13 epitopes and assess staining pattern similarity

  • Cross-species reactivity: If INTS13 is conserved across species, evaluate appropriate cross-reactivity patterns

  • Isotype control experiments: Use FITC-conjugated isotype control antibodies to establish background staining levels

• Biological validation strategies:

  • Expression pattern analysis: Verify that INTS13 expression patterns match expected biological distribution

  • Cell cycle analysis: Confirm that INTS13 expression/localization changes during cell cycle match literature reports

  • Co-localization studies: Demonstrate appropriate co-localization with known INTS13 interacting proteins

  • Functional correlation: Show correlation between INTS13 staining patterns and known functional outcomes

• Documentation and reporting standards:

  Validation Element	Required Information	Purpose
  Antibody details	Clone, lot number, manufacturer, concentration	Reproducibility
  Positive controls	Cell lines or tissues with known INTS13 expression	Confirm detection capability
  Negative controls	INTS13-negative samples or knockout models	Establish specificity
  Protocol parameters	Fixation, permeabilization, dilution, incubation details	Methodological transparency
  Alternative methods	Correlation with orthogonal detection techniques	Multi-modal validation

By implementing this multi-faceted validation strategy, researchers can establish high confidence in the specificity of FITC-conjugated INTS13 antibody staining in novel experimental systems, ensuring reliable and reproducible research outcomes.

How might advances in fluorophore technology impact the applications of INTS13 antibodies beyond current FITC conjugation limitations?

The landscape of fluorophore technology is rapidly evolving, offering numerous opportunities to enhance INTS13 antibody applications beyond the limitations of conventional FITC conjugation. Future research directions include:

• Next-generation fluorophores:

  • Quantum dots: Provide exceptional photostability, narrow emission spectra, and size-tunable fluorescence properties for long-term INTS13 tracking

  • Fluorescent proteins: Direct genetic fusion of fluorescent proteins to INTS13 for live-cell imaging without antibody-mediated detection

  • Reversibly switchable fluorophores: Enable super-resolution microscopy applications for nanoscale visualization of INTS13 distribution

  • Two-photon excitable fluorophores: Allow deeper tissue penetration with reduced phototoxicity for in vivo INTS13 studies

• Emerging methodological approaches:

  • Proximity ligation assays: Visualize INTS13 protein-protein interactions with single-molecule sensitivity

  • Fluorescence lifetime imaging (FLIM): Detect changes in INTS13 microenvironment independent of concentration variations

  • Förster resonance energy transfer (FRET): Measure dynamic INTS13 interactions with binding partners in living cells

  • Correlative light and electron microscopy (CLEM): Combine fluorescence localization of INTS13 with ultrastructural context

• Anticipated technological developments:

  Technology	Projected Timeline	Potential Impact on INTS13 Research
  Super-resolution antibody imaging	Current-3 years	Nanoscale mapping of INTS13 within nuclear substructures
  Biodegradable quantum dots	3-5 years	Reduced toxicity for long-term INTS13 tracking in live models
  Photoswitchable organic dyes	1-3 years	Enhanced multiplexing capabilities for complex INTS13 interaction networks
  Machine learning image analysis	Current-2 years	Automated quantification of subtle INTS13 distribution patterns
  Infrared fluorophores	2-4 years	Deeper tissue imaging of INTS13 in complex tissue environments

• Integration with emerging single-cell technologies:

  • Mass cytometry (CyTOF) with metal-tagged antibodies instead of fluorophores

  • Spatial transcriptomics combined with INTS13 protein detection

  • Single-cell proteomics correlated with INTS13 antibody-based imaging

  • Live-cell tracking of INTS13 dynamics during cell cycle progression

These advancing technologies will likely transform our ability to study INTS13 biology by providing enhanced spatial resolution, improved sensitivity, expanded multiplexing capabilities, and integration with multi-omics approaches, ultimately leading to more comprehensive understanding of INTS13's functional roles in cellular processes.