INTS3 Antibody

What is INTS3 and why is it significant in research?

INTS3 (Integrator Complex Subunit 3) is a critical component of the integrator complex that interacts with RNA polymerase II (RNAPII) C-terminal domain and participates in the processing of small nuclear RNAs (snRNAs) . Additionally, INTS3 functions as a sensor of single-strand DNA (SOSS-A) and plays important roles in mRNA processing and transcription regulation . With a molecular weight of approximately 118.1 kDa, INTS3 has gained significant research interest due to its overexpression in certain cancer types, including colorectal cancer, making it a potential biomarker for disease detection and therapeutic targeting .

The significance of INTS3 in research extends beyond its normal cellular functions. Its involvement in fundamental cellular processes makes it a valuable target for studying transcriptional regulation, DNA damage response pathways, and oncogenic mechanisms. Researchers typically select INTS3 antibodies based on their experimental needs, considering factors such as specificity, sensitivity, and compatibility with specific applications.

What are the key differences between polyclonal and monoclonal INTS3 antibodies?

Polyclonal INTS3 antibodies, such as the rabbit polyclonal antibody (16620-1-AP), recognize multiple epitopes of the INTS3 protein, potentially providing higher sensitivity but with increased risk of cross-reactivity . These antibodies are typically generated by immunizing rabbits with INTS3 fusion proteins or specific peptide sequences. In contrast, monoclonal antibodies (like the 4C12 clone) recognize a single epitope, offering higher specificity but potentially lower sensitivity than polyclonal options .

When selecting between these antibody types, researchers should consider:

How should researchers evaluate and validate INTS3 antibody specificity?

Antibody validation is crucial for generating reliable research data. For INTS3 antibodies, a multi-tiered validation approach is recommended:

• Genetic validation: Test antibody reactivity in INTS3 knockdown/knockout samples versus controls. Specific INTS3 antibodies should show significantly reduced or absent signal in knockout samples.

• Molecular weight verification: Confirm that the observed molecular weight matches the expected 118 kDa of INTS3 protein in Western blot applications .

• Cross-validation: Compare results using multiple INTS3 antibodies targeting different epitopes.

• Orthogonal validation: Correlate antibody-based detection with other methods such as mass spectrometry or RNA expression.

• Application-specific controls: Include appropriate positive controls (HEK-293 or HeLa cells have been validated for many INTS3 antibodies) and negative controls for each application .

The most common pitfall in INTS3 research is inadequate validation, leading to misinterpretation of results, particularly in complex sample types or when studying INTS3 interaction partners.

What are the optimal protocols for using INTS3 antibodies in Western blotting?

Western blotting is one of the most common applications for INTS3 antibodies. Based on validated protocols, researchers should consider:

Sample preparation:

• Use RIPA or NP-40 buffer with protease inhibitors for effective INTS3 extraction

• Include phosphatase inhibitors if phosphorylated forms are of interest

• Sonicate briefly to shear DNA and improve protein release

Western blot parameters:

• Load 20-40 μg of total protein per lane for cell lysates

• Use 7.5-10% polyacrylamide gels to properly resolve the 118 kDa INTS3 protein

• Transfer to PVDF membranes at 100V for 90 minutes or 30V overnight at 4°C

Antibody dilutions and detection:

• For rabbit polyclonal INTS3 antibodies: use 1:1000-1:4000 dilution range

• For monoclonal INTS3 antibodies: follow manufacturer-specific recommendations

• Longer primary antibody incubation (overnight at 4°C) typically yields better results

• Secondary antibody incubation at 1:5000-1:10000 for 1 hour at room temperature

HEK-293 and HeLa cells serve as excellent positive controls as they have been validated to express detectable INTS3 levels .

How can researchers optimize INTS3 immunoprecipitation experiments?

Immunoprecipitation (IP) is critical for studying INTS3 protein interactions and modifications. Optimized protocols include:

Pre-clearing strategy:

• Pre-clear lysates with protein A/G beads for 1 hour at 4°C to reduce non-specific binding

• Use 1-3 mg of total protein for sufficient INTS3 pulldown

Antibody amount:

• For optimal INTS3 IP, use 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate

• Pre-form antibody-bead complexes before adding lysate for reduced background

Washing conditions:

• Use increasingly stringent washes (TBS-T followed by higher salt buffers)

• Include at least 4-5 wash steps to minimize non-specific binding

Elution techniques:

• For interaction studies: gentle elution with peptide competition

• For downstream analysis: more stringent SDS-based elution

When analyzing INTS3 complex formation, gentler lysis conditions may help preserve protein-protein interactions that could be disrupted by harsher detergents.

What are the critical considerations for INTS3 immunohistochemistry?

Immunohistochemistry (IHC) allows visualization of INTS3 expression in tissue contexts. Important methodology factors include:

Antigen retrieval:

• Heat-induced epitope retrieval (HIER) with TE buffer at pH 9.0 is recommended for INTS3

• Alternatively, citrate buffer at pH 6.0 can be used with optimized heating time

• Pressure cooking for 3-5 minutes often yields superior results compared to water bath methods

Antibody dilution and incubation:

• Start with 1:200-1:1000 dilution range for INTS3 antibodies in IHC

• Optimize by testing multiple dilutions on known positive controls

• Incubate primary antibody overnight at 4°C for best signal-to-noise ratio

Detection systems:

• Polymer-based detection systems typically provide better sensitivity for INTS3 visualization

• DAB substrate development should be carefully timed and monitored microscopically

Human ovarian cancer tissue has been validated as a positive control for INTS3 IHC . Researchers should include appropriate negative controls (primary antibody omission, isotype controls) to confirm specificity.

How does INTS3 function in DNA damage response pathways?

INTS3 functions as a component of the SOSS (Sensor Of Single-Strand DNA) complex, playing a critical role in the DNA damage response pathway. When studying this function, researchers should consider:

Experimental design elements:

• Induce DNA damage using UV, ionizing radiation, or chemical agents (e.g., etoposide)

• Monitor INTS3 localization to DNA damage sites via immunofluorescence microscopy

• Assess temporal dynamics of INTS3 recruitment following damage (typically peaks at 2-6 hours)

Methodological considerations:

• Co-staining with γH2AX confirms INTS3 localization to DNA damage foci

• ChIP experiments can assess INTS3 binding to damaged chromatin regions

• Co-immunoprecipitation with other SOSS complex members (SOSSB1/2, SOSSC) confirms complex integrity

Advanced analytical approaches:

• Proximity ligation assays (PLA) can verify INTS3 interactions with repair proteins in situ

• CRISPR-mediated tagging of endogenous INTS3 for live-cell imaging avoids artifacts from overexpression

These methodologies have revealed that INTS3 is essential for proper ATM/ATR signaling and homologous recombination repair pathway activation after DNA damage.

What methodologies are most effective for studying INTS3 in cancer research?

INTS3 has been implicated in cancer biology, particularly in colorectal cancer where it shows overexpression . Recommended research approaches include:

Expression analysis methods:

• Use IHC with properly validated INTS3 antibodies (1:200-1:1000 dilution) on tissue microarrays

• Quantify expression using digital pathology tools with appropriate controls

• Correlate with clinical parameters and survival data

Functional studies:

• CRISPR/Cas9 knockout or siRNA knockdown in cancer cell lines

• Rescue experiments with wild-type vs. mutant INTS3

• Proliferation, migration, and invasion assays following INTS3 modulation

Biomarker validation approach:

• Multi-cohort analysis with consistent IHC protocols

• Receiver operating characteristic (ROC) analysis to determine optimal cutoff values

• Multi-variable analysis controlling for standard clinicopathological parameters

These studies should include appropriate cell line models that represent the cancer type of interest, with HeLa cells serving as a reliable positive control for INTS3 expression .

How can researchers effectively use INTS3 antibodies in ChIP experiments?

Chromatin immunoprecipitation (ChIP) allows investigation of INTS3's role in transcriptional regulation. Optimized protocols include:

Crosslinking optimization:

• Standard 1% formaldehyde for 10 minutes at room temperature

• Dual crosslinking with DSG followed by formaldehyde may improve recovery of INTS3 complexes

• Quench with 125mM glycine for 5 minutes

Sonication parameters:

• Optimize sonication to achieve 200-500bp DNA fragments

• Verify fragmentation efficiency via gel electrophoresis before proceeding

Immunoprecipitation conditions:

• Use 2-5μg of ChIP-grade INTS3 antibody per reaction

• Include IgG control and positive control antibody (e.g., RNA Pol II)

• Increase wash stringency gradually to reduce background

Analysis approaches:

• qPCR for known INTS3-associated regions

• ChIP-seq for genome-wide binding profile

• Integration with RNA-seq data to correlate binding with expression changes

The ChIP protocol should be validated using primers targeting regions where INTS3 is known to bind, particularly near genes involved in snRNA processing or at sites of active transcription.

How can researchers address non-specific binding issues with INTS3 antibodies?

Non-specific binding is a common challenge when working with INTS3 antibodies. Effective troubleshooting strategies include:

Western blot optimization:

• Increase blocking stringency (5% BSA or milk in TBS-T for 1-2 hours)

• Use graduated washing steps with increasing detergent concentration

• Titrate primary antibody; for INTS3, start at 1:1000 and adjust based on results

• Increase the number and duration of wash steps (minimum 3×5 minutes with TBS-T)

Immunoprecipitation refinement:

• Pre-clear lysates thoroughly using protein A/G beads

• Include competing proteins (BSA) in wash buffers

• Consider epitope-tagged INTS3 with tag-specific antibodies for cleaner results

Immunohistochemistry improvements:

• Extend blocking time to 1 hour with serum from the secondary antibody species

• Include 0.1-0.3% Triton X-100 in antibody diluent to reduce hydrophobic interactions

• Use more dilute antibody with longer incubation times (e.g., 1:1000 overnight at 4°C)

When persistent background occurs, peptide competition assays can determine if binding is specific to the INTS3 epitope or represents non-specific interactions.

What strategies help optimize signal detection in INTS3 immunofluorescence?

Immunofluorescence with INTS3 antibodies requires careful optimization to visualize this nuclear protein effectively:

Fixation and permeabilization:

• Compare paraformaldehyde (4%) vs. methanol fixation

• Test different permeabilization methods (0.1-0.5% Triton X-100 for 10-15 minutes)

• Evaluate epitope accessibility with heat-mediated antigen retrieval

Signal amplification options:

• Tyramide signal amplification for low abundance detection

• Sequential application of primary and secondary antibodies with washing

• Optimized secondary antibody concentration (typically 1:500-1:1000)

Imaging parameters:

• Z-stack acquisition to capture the full nuclear volume

• Deconvolution to improve signal-to-noise ratio

• Consistent exposure settings across experimental conditions

Counterstaining strategy:

• DAPI for nuclear visualization (INTS3 is predominantly nuclear)

• Use of other organelle markers to assess potential cytoplasmic localization

• Co-staining with known INTS3 interactors to confirm specific localization

Optimization typically requires testing multiple conditions systematically, with HeLa cells serving as reliable positive controls for INTS3 immunofluorescence .

How should researchers interpret conflicting INTS3 antibody results?

When different INTS3 antibodies yield contradictory results, systematic investigation is necessary:

Validation comparison:

• Review validation data for each antibody (knockout controls, overexpression systems)

• Consider epitope locations and potential masking in protein complexes

• Evaluate species cross-reactivity if working with non-human samples

Technical variables:

• Standardize sample preparation methods across experiments

• Ensure identical protein amounts are used for direct comparisons

• Document lot numbers of antibodies as batch variation can occur

Resolution strategies:

• Employ orthogonal detection methods (mass spectrometry, RNA expression)

• Use tagged INTS3 constructs with anti-tag antibodies as references

• Consult literature for previously validated INTS3 antibody applications

When publishing, researchers should report detailed antibody information, including catalog numbers, dilutions, and validation methods to ensure reproducibility.

How can INTS3 antibodies be utilized in studying RNA processing mechanisms?

INTS3 is part of the integrator complex involved in the processing of small nuclear RNAs (snRNAs) . Researchers investigating this function should consider:

Experimental approaches:

• RNA immunoprecipitation (RIP) using validated INTS3 antibodies

• CLIP-seq (crosslinking immunoprecipitation) to identify direct RNA binding sites

• Pulse-chase labeling of nascent RNA followed by INTS3 immunoprecipitation

Analytical methodologies:

• Integration of RIP-seq with RNA-seq data to correlate binding with processing outcomes

• Structure-function analysis with domain-specific antibodies

• Co-localization studies with other integrator complex components

These approaches can reveal INTS3's role in coordinating transcription termination with RNA processing, particularly for non-coding RNAs and enhancer RNAs that may influence gene expression programs.

What considerations are important when studying INTS3 as a potential biomarker?

INTS3 has been identified as overexpressed in certain tumors, suggesting potential as a diagnostic or prognostic biomarker . Key research considerations include:

Standardization requirements:

• Establish validated IHC protocols with specific cutoff values

• Coordinate antibody selection and dilution across research centers (1:200-1:1000 range)

• Document pre-analytical variables (fixation time, processing methods)

Validation framework:

• Training and validation cohorts with sufficient statistical power

• Multivariate analysis controlling for established prognostic factors

• Comparison with current gold standard biomarkers

Clinical utility assessment:

• Correlation with therapy response in retrospective cohorts

• Integration with other molecular markers in multiplexed approaches

• Evaluation in minimally invasive samples (liquid biopsies, circulating tumor cells)

Research in this area should focus on reproducibility across laboratories and clinical relevance rather than mere statistical associations.