INTS10 Antibody, FITC conjugated

What is INTS10 and why is it important in research applications?

INTS10 (also known as C8orf35 or INT10) is a critical 710 amino acid protein that functions as a subunit of the Integrator complex. This complex associates with the C-terminal domain of RNA polymerase II large subunit and plays an essential role in mediating 3'-end processing of small nuclear RNAs, particularly U1 . INTS10 is primarily localized to the nucleus and contributes to fundamental RNA processing mechanisms .

The importance of INTS10 in research extends beyond basic RNA biology. Recent studies have revealed that the Integrator complex participates in transcriptional regulation through gene-specific mechanisms. INTS10 forms part of a stable module with INTS13 and INTS14, which has been identified as the INT transcription factor binding module (ITFM) . This module provides a platform for sequence-specific transcription factors and transcription effector complexes to bind, influencing transcription decisions between productive elongation and abortive early termination .

What detection methods are most effective when using INTS10 antibodies?

Based on validated research applications, INTS10 antibodies have demonstrated effectiveness in several detection methods:

• Western Blotting (WB): Rabbit polyclonal antibodies to INTS10 have been validated for detecting endogenous levels of total INTS10 protein in human samples. Optimal dilutions of approximately 1/475 have been effective when using 40μg of total protein lysate from cell lines like HeLa and HepG2 .

• Immunohistochemistry (IHC): INTS10 antibodies can be used for detection in paraffin-embedded tissue sections, with successful applications documented in human thyroid cancer tissue at a dilution of 1/50 .

• Immunofluorescence (IF): Polyclonal antibodies against INTS10 have been verified for immunofluorescence applications, with recommended dilutions of 1:50-1:200 successfully tested in cell lines such as A549 .

For optimal results, researchers should consider specific sample preparation protocols for each technique and verify antibody reactivity with their species of interest, as most validated INTS10 antibodies have been tested for human and sometimes mouse reactivity .

What are the best sample preparation methods for INTS10 detection?

Effective sample preparation is critical for successful INTS10 detection:

For Western Blotting:

• Cell lysate preparation should include proper lysis buffers containing protease inhibitors

• Recommended protein loading of 40μg per lane has been validated in published protocols

• For gel separation, 6% SDS-PAGE has been successfully used for INTS10 detection

• Proteins should be completely transferred to membranes (typically PVDF or nitrocellulose)

• Blocking with appropriate buffers (usually 5% non-fat milk or BSA) is essential to minimize background

For Immunohistochemistry:

• Paraffin-embedded tissue sections should undergo proper antigen retrieval

• Fixation methods must preserve the INTS10 epitope structure

• Background reduction techniques should be employed, particularly when working with tissues with high endogenous peroxidase activity

• Sections of approximately 5μm thickness are typically suitable

For Immunofluorescence:

• Cells should be fixed with paraformaldehyde and permeabilized appropriately

• Blocking with serum matching the secondary antibody host species improves specificity

• Careful washing steps are essential to minimize background fluorescence

• Mounting media containing DAPI for nuclear counterstaining helps confirm the nuclear localization of INTS10

What are the advantages of using FITC-conjugated INTS10 antibodies versus unconjugated primary antibodies?

FITC-conjugated INTS10 antibodies offer several distinct advantages in research applications:

Direct Detection Benefits:

• Elimination of secondary antibody steps, reducing protocol time and complexity

• Minimization of potential cross-reactivity issues that can occur with secondary antibodies

• Reduction in background signal often associated with two-step detection methods

• Enabling of multiplexing with antibodies raised in the same host species

Methodological Considerations:

• Direct conjugation maintains a 1:1 ratio of fluorophore to antibody, potentially leading to more quantitative results

• Single-step protocols reduce washing steps where signal loss might occur

• Particularly valuable for co-localization studies with other proteins

While direct FITC-conjugated INTS10 antibodies offer these advantages, researchers sometimes opt for a primary-secondary approach using unconjugated INTS10 antibodies with FITC-conjugated secondary antibodies (such as Goat Anti-Rabbit IgG H&L Antibody with FITC conjugation ). This approach offers signal amplification, as multiple secondary antibodies can bind each primary antibody, enhancing detection sensitivity for low-abundance proteins. The choice between direct and indirect detection should be guided by experimental requirements for sensitivity versus specificity.

How can researchers optimize FITC-conjugated antibody signals when studying INTS10?

Optimization of FITC-conjugated antibody signals requires attention to several technical aspects:

Protocol Optimization:

• Concentration titration: Begin with manufacturer-recommended dilutions (typically 1:50-1:200 for IF applications) and adjust based on signal-to-noise ratio

• Incubation conditions: Optimize both temperature (4°C, room temperature) and duration (1-24 hours)

• Mounting media selection: Use anti-fade mounting media specifically formulated to preserve FITC fluorescence

• Fixation method evaluation: Compare paraformaldehyde, methanol, and acetone fixation effects on epitope accessibility and signal strength

Signal Preservation Techniques:

• Minimize exposure to light during all protocol steps

• Store slides at 4°C in the dark and image within 1-2 days of preparation

• Consider photobleaching controls in experimental design

• When necessary, utilize tyramide signal amplification systems to enhance FITC signal while maintaining specificity

Imaging Considerations:

• Use appropriate excitation (approximately 495nm) and emission (approximately 520nm) filter sets

• Optimize exposure settings to prevent photobleaching during image acquisition

• Consider confocal microscopy for improved signal-to-noise ratio, particularly when examining nuclear localization of INTS10

• Implement consistent image acquisition parameters across experimental conditions

How can researchers validate the specificity of INTS10 antibodies for research applications?

Rigorous validation of INTS10 antibody specificity is essential for meaningful research outcomes:

Recommended Validation Approaches:

• siRNA or CRISPR/Cas9 knockdown controls: Confirmation that the INTS10 signal decreases following gene silencing or knockout provides strong validation of antibody specificity . Researchers have successfully used siRNA approaches to study INTS family genes, which could be adapted for INTS10-specific validation.

• Western blot analysis: Verification that the antibody detects a single band of the expected molecular weight (~77-80 kDa for INTS10) in target samples. Published validation data shows successful detection in HeLa and HepG2 cell lysates .

• Recombinant protein controls: Using purified recombinant INTS10 protein as a positive control and comparing band patterns with endogenous samples.

• Immunoprecipitation followed by mass spectrometry: Confirming that INTS10 is among the identified proteins after pull-down with the antibody. Studies have successfully used this approach with INTS10, identifying it as part of the INTS10/13/14 module .

• Cross-validation with multiple antibodies: Using antibodies targeting different epitopes of INTS10 to confirm consistent localization patterns. Research has employed antibodies against different regions of related proteins (like INTS13) to verify results .

• Peptide competition assays: Pre-incubation of the antibody with immunizing peptide should abolish specific signal if the antibody is truly specific.

What are the methodological challenges in studying INTS10 interactions with other Integrator complex components?

Investigating INTS10 interactions presents several methodological challenges:

Technical Challenges and Solutions:

• Complex stability issues: The Integrator complex consists of 14+ subunits, making isolation of intact complexes difficult. Researchers have overcome this by focusing on stable subcomplexes like the INTS10/13/14 module using tandem affinity purification approaches .

• Interaction detection limitations: Standard yeast two-hybrid systems may fail to detect interactions between individual Integrator subunits, as observed with INTS13 . This suggests INTS10 interactions may depend on multi-protein interfaces rather than binary interactions.

• Nuclear localization complexities: As a nuclear protein, studying INTS10 interactions requires careful cell fractionation or in situ approaches. Nuclear extraction protocols must balance efficiency with preservation of protein-protein interactions.

• Co-immunoprecipitation optimization: Successful co-IP of INTS10 with partners like INTS13 and INTS14 requires optimization of buffer conditions, including salt concentration, detergent types, and incubation times.

• Recombinant expression challenges: Reconstituting functional INTS10-containing complexes in vitro requires co-expression systems, often using insect cells. Size-exclusion chromatography has been effective for confirming the formation of stable complexes .

• Visualization of interactions: Advanced microscopy techniques like proximity ligation assay (PLA) or FRET may be necessary to visualize INTS10 interactions in situ, particularly given its nuclear localization.

How can INTS10 antibodies contribute to understanding transcription regulation mechanisms?

INTS10 antibodies serve as powerful tools for investigating transcriptional regulatory mechanisms:

Recent research has revealed that the INTS10-13-14 module forms an Integrator TF binding module (ITFM) that directly interacts with sequence-specific transcription factors and transcription effector complexes . This discovery positions INTS10 at the intersection of transcription and RNA processing, making antibodies against this protein valuable for studying:

• Chromatin Immunoprecipitation (ChIP) applications: INTS10 antibodies can be used to map genomic binding sites, revealing promoter-proximal pause sites where the Integrator complex influences transcription decisions .

• Co-immunoprecipitation studies: Identifying novel protein interactions with transcription factors like ZNF608, ZNF609, ZNF655, and ZNF687, which have been shown to co-purify with INTS10 .

• Stress response investigations: As the TF-interactome of Integrator is modulated by stress conditions, INTS10 antibodies can help track changes in complex composition and chromatin localization under different cellular stresses .

• Functional analysis of ciliopathies: Studies have linked Integrator components to ciliopathy disorders, and INTS10 antibodies may help investigate these connections through immunofluorescence studies of primary cilia formation .

• Multi-omics approaches: Combining INTS10 ChIP-seq with RNA-seq to correlate binding sites with transcriptional outcomes, particularly at genes regulated through promoter-proximal pausing mechanisms.

What are the most common technical issues when using FITC-conjugated antibodies for INTS10 detection?

Researchers frequently encounter several challenges when using FITC-conjugated antibodies for INTS10 detection:

Issue 1: Photobleaching and Signal Fading

• FITC is relatively susceptible to photobleaching compared to other fluorophores

• Solution: Use anti-fade mounting media specifically designed for FITC preservation; minimize exposure to light during all protocol steps; consider imaging slides within 24-48 hours of preparation

Issue 2: Autofluorescence Interference

• Cellular components, particularly in certain tissues, can generate autofluorescence in the FITC emission range

• Solution: Include appropriate autofluorescence controls; consider spectral unmixing during image acquisition; use Sudan Black B treatment to reduce autofluorescence in tissues

Issue 3: pH Sensitivity

• FITC fluorescence is sensitive to pH variations, with optimal emission at slightly alkaline pH

• Solution: Maintain consistent buffer pH (ideally pH 7.4-8.0) during antibody incubation and washing steps; use pH-stabilized mounting media

Issue 4: Low Signal-to-Noise Ratio

• Nuclear proteins like INTS10 can sometimes present detection challenges due to high background

• Solution: Optimize blocking conditions (duration, composition); increase washing stringency; titrate antibody concentration carefully; consider signal amplification systems if necessary

Issue 5: Non-specific Binding

• Direct conjugates can sometimes exhibit higher non-specific binding

• Solution: Include blocking peptides specific to the FITC-conjugated INTS10 antibody; optimize blocking with sera matched to the host species; implement more stringent washing protocols

How can researchers quantitatively analyze INTS10 expression or localization data?

Quantitative analysis of INTS10 expression or localization requires rigorous methodological approaches:

For Western Blot Quantification:

• Use proper loading controls (housekeeping proteins appropriate for nuclear proteins)

• Implement linear range detection methods to ensure measurements fall within quantifiable range

• Apply densitometry software (ImageJ, Image Lab, etc.) with background subtraction

• Normalize INTS10 signal to loading controls for each sample

• Present data as fold change relative to control conditions

For Immunofluorescence Quantification:

• Nuclear Intensity Measurement:

  • Define nuclear regions using DAPI counterstain

  • Measure mean fluorescence intensity of INTS10 signal within nuclear boundaries

  • Subtract background from regions adjacent to nuclei

  • Compare nuclear INTS10 intensities across experimental conditions

• Co-localization Analysis:

  • When studying INTS10 interaction with other proteins, use co-localization coefficients (Pearson's, Manders')

  • Implement appropriate controls for fluorescence bleed-through

  • Consider super-resolution microscopy for detailed co-localization studies

• Distribution Pattern Analysis:

  • Quantify the distribution pattern of INTS10 within nuclei (e.g., nucleoplasmic vs. nucleolar)

  • Create intensity line profiles across nuclei to visualize distribution patterns

  • Use granularity or texture analysis to quantify changes in nuclear distribution patterns

Statistical Approach:

• Analyze sufficient cell numbers (typically n>50 cells per condition)

• Apply appropriate statistical tests (t-test, ANOVA) based on data distribution

• Present results with error bars (standard deviation or standard error) and significance indicators

How can INTS10 antibodies be used to investigate the relationship between transcription and RNA processing?

INTS10 antibodies provide valuable tools for investigating the nexus between transcription and RNA processing:

The Integrator complex, of which INTS10 is a component, plays a dual role in transcription attenuation and snRNA processing. The discovery that the INTS10-13-14 module directly binds transcription factors positions INTS10 as a critical link between these processes . Several methodological approaches can leverage INTS10 antibodies to explore this relationship:

• Chromatin Immunoprecipitation sequencing (ChIP-seq):

  • Map genome-wide binding sites of INTS10 to identify genes regulated by Integrator

  • Compare INTS10 binding patterns with RNA Polymerase II occupancy profiles

  • Integrate with nascent RNA sequencing data to correlate binding with transcriptional outcomes

• Proximity-based proteomic approaches:

  • Implement BioID or APEX2 proximity labeling with INTS10 as bait to identify proteins in its vicinity

  • Compare interactome changes under different transcriptional states or stress conditions

  • Correlate with known transcription factor binding sites at Integrator-regulated genes

• Functional analysis using reporter systems:

  • Combine INTS10 antibody-based imaging with MS2-tagged nascent RNA visualization

  • Monitor co-localization of INTS10 with nascent transcripts in real-time

  • Correlate INTS10 recruitment with transcriptional pause release or termination events

• Stress response studies:

  • Track changes in INTS10 localization and interaction networks under conditions that modulate transcription

  • Investigate how stress-induced transcription factors alter their association with the INTS10-13-14 module

  • Correlate with changes in promoter-proximal pausing and productive elongation