INTS6 Antibody, HRP conjugated

What is INTS6 and why is it significant in molecular research?

INTS6 (Integrator complex subunit 6) is a component of the Integrator (INT) complex involved in the transcription and processing of small nuclear RNAs (snRNA) U1 and U2. The Integrator complex associates with the C-terminal domain (CTD) of RNA polymerase II largest subunit (POLR2A) and is recruited to the U1 and U2 snRNAs genes . INTS6 has gained significant research interest due to its potential tumor suppressor activity, with studies showing that its ectopic expression can suppress tumor cell growth .

INTS6 is also known by several alternative names including DBI1, DDX26, DDX26A, Int6, DBI-1, Protein DDX26, Protein deleted in cancer 1, and DICE1 . Beyond RNA processing, it mediates recruitment of cytoplasmic dynein to the nuclear envelope, likely functioning as a component of the INT complex .

Recent research in hepatocellular carcinoma (HCC) has revealed that INTS6 and its pseudogene INTS6P1 function as tumor suppressors through a novel regulatory mechanism involving competition for oncomiR-17-5p . Expression studies have demonstrated that both INTS6 and INTS6P1 are down-regulated in approximately 70% of HCC cases compared to normal liver tissues, with their expression levels showing positive correlation .

How does HRP conjugation work in antibodies and what advantages does it offer?

HRP (Horseradish peroxidase) conjugation creates a direct detection system that enhances research applications of antibodies. HRP is a 44 kDa glycoprotein containing 6 lysine residues that can be covalently linked to antibodies . When HRP is conjugated to INTS6 antibodies, it enables detection through enzymatic amplification of signal.

The HRP enzyme catalyzes chromogenic reactions that produce visible signals. The most common substrate system involves diaminobenzidine (DAB), which in the presence of hydrogen peroxide (H₂O₂) is converted into a water-insoluble brown precipitate that can be visualized under standard light microscopy. Alternative substrates include ABTS, TMB, and TMBUS, each offering different visualization properties .

HRP conjugation offers several methodological advantages:

• Direct detection eliminates the need for secondary antibody incubation steps, which reduces protocol time and can decrease non-specific background signal.

• It avoids potential cross-species reactivity issues that can complicate indirect detection systems.

• Signal amplification through enzymatic activity improves sensitivity compared to direct fluorescent conjugates.

• The stable chromogenic products allow for long-term sample storage and analysis, unlike fluorescent methods that may fade over time .

What research applications are optimal for INTS6 Antibody, HRP conjugated?

INTS6 Antibody, HRP conjugated can be employed in multiple research methodologies with specific technical considerations for each:

Western Blotting Applications:
The direct HRP conjugation enables efficient detection of INTS6 in protein lysates separated by electrophoresis . For optimal results, researchers should load 20-50 μg of total protein per lane and employ enhanced chemiluminescence detection systems. This approach allows quantitative comparison of INTS6 expression between experimental conditions or between normal and pathological samples.

Immunohistochemistry (IHC):
HRP-conjugated INTS6 antibodies have been successfully applied to formalin-fixed, paraffin-embedded (FFPE) tissue sections . Published protocols indicate effective staining at dilutions of 1/200 (1μg/ml) in human carcinoma tissues . Antigen retrieval methods, typically involving citrate buffer and heat treatment, are essential for optimal epitope exposure in fixed tissues.

ELISA (Enzyme-Linked Immunosorbent Assay):
The HRP conjugation makes these antibodies particularly suitable for ELISA applications where direct detection improves assay efficiency . This methodology allows quantitative assessment of INTS6 protein levels in complex biological samples including cell lysates, tissue extracts, and potentially biological fluids.

Cell-Based Assays:
These antibodies can effectively detect INTS6 in cultured cells, including cancer cell lines like Huh7, MHCC97H, MHCC97L, and HepG2, which have been shown to express lower levels of INTS6 compared to normal human hepatocytes . This application is valuable for monitoring expression changes following experimental manipulations.

What are the critical factors for storage and handling to preserve antibody functionality?

Maintaining optimal functionality of INTS6 Antibody, HRP conjugated requires careful attention to storage and handling procedures:

Storage Parameters:

• Long-term storage should be at -20°C or -80°C, with -80°C recommended for extended preservation .

• Avoid repeated freeze-thaw cycles which can degrade both the antibody and the conjugated HRP enzyme .

• Commercial preparations typically contain stabilizers such as 50% glycerol and preservatives like 0.03% Proclin 300 in 0.01M PBS (pH 7.4) buffer .

Handling Protocol:

• Thaw aliquots slowly on ice or at 4°C rather than at room temperature to minimize protein denaturation.

• Prepare working aliquots of appropriate volumes during initial thawing to avoid repeated freezing of the stock solution.

• Maintain cold chain during experimental procedures; keep antibody on ice when in use.

• Return unused portions to appropriate storage conditions immediately after use.

• For dilution preparations, use high-quality, nuclease-free buffers to prevent contamination.

Stability Considerations:

• HRP conjugation can affect antibody stability differently than unconjugated antibodies, generally making them more sensitive to storage conditions.

• The enzymatic activity of HRP may decrease more rapidly than antibody binding capacity, potentially resulting in reduced signal over time.

• Preservatives in commercial formulations help maintain stability but cannot completely prevent degradation under suboptimal conditions.

• Monitor lot-to-lot variation and establish internal validation protocols when using new lots.

Following these storage and handling guidelines will help ensure consistent experimental results when working with INTS6 Antibody, HRP conjugated.

What methodological approaches are essential when using INTS6 Antibody, HRP conjugated in tumor suppression studies?

Investigating INTS6's tumor suppressor function requires sophisticated experimental design and careful methodological considerations:

Sample Preparation Methodology:

• For tissue microarrays or individual FFPE sections, standardize fixation time (24 hours in 10% neutral buffered formalin) and processing parameters to maintain consistent antigen preservation .

• Implement rigorous antigen retrieval optimization; heat-induced epitope retrieval using citrate buffer (pH 6.0) has proven effective for preserving INTS6 epitopes in fixed tissues.

• Develop a blocking strategy that addresses both non-specific antibody binding (protein blocking) and endogenous peroxidase activity (H₂O₂ treatment) to maximize signal-to-noise ratio.

• For cell line models, standardize lysis conditions using RIPA buffer supplemented with protease inhibitors to prevent INTS6 degradation during extraction.

Experimental Controls Framework:

• Include positive controls (normal liver tissue) where INTS6 expression is documented to be higher than in HCC tissues .

• Implement antibody validation through siRNA-mediated knockdown of INTS6, which should result in corresponding reduction of antibody signal .

• Consider epitope availability; some INTS6 antibodies target synthetic peptides within amino acids 750-800 , which may be affected by protein conformation or post-translational modifications.

Integrated Functional Analysis:

• Combine protein detection with functional assays as demonstrated in published research: siRNA knockdown of INTS6 increased cell growth in HCC lines, while overexpression induced growth arrest .

• Incorporate cell death and migration assays (such as scratch assays) to comprehensively assess tumor suppressive properties .

• Design experiments that analyze both INTS6 and its pseudogene INTS6P1 simultaneously to capture their reciprocal regulation .

Quantitative Assessment Protocol:

• For IHC, develop a standardized scoring system that accounts for both staining intensity (0-3) and percentage of positive cells (0-100%) to generate H-scores (0-300).

• When performing western blot analysis, normalize INTS6 signal to established housekeeping proteins like β-actin or GAPDH, and use digital imaging systems with validated dynamic range.

• For comparative studies across multiple samples, implement batch controls and inter-run calibrators to minimize technical variation.

These methodological approaches will enhance experimental rigor when investigating INTS6's tumor suppressor functions using HRP-conjugated antibodies.

How should researchers design experiments to investigate the regulatory relationship between INTS6 and its pseudogene INTS6P1?

Investigating the complex regulatory relationship between INTS6 and INTS6P1 requires a multi-faceted experimental approach:

Differential Detection Strategy:

• Employ HRP-conjugated INTS6 antibodies for protein detection while establishing transcript-specific qRT-PCR protocols that can distinguish between INTS6 and INTS6P1 despite their 96% sequence homology .

• Design primer sets targeting unique regions in each transcript, with particular attention to the 3' untranslated regions which typically show greater divergence.

• Validate primer specificity using synthetic templates and establish standard curves for accurate quantification.

Mechanistic Investigation Model:

• Implement a sequential experimental design to test the competing endogenous RNA hypothesis:

  • Perform siRNA-mediated knockdown of INTS6P1 and measure effects on INTS6 protein levels using HRP-conjugated antibodies

  • Overexpress INTS6P1 and quantify changes in INTS6 protein expression

  • Manipulate miR-17-5p levels through mimics or inhibitors and assess impact on both INTS6 and INTS6P1

  • Use luciferase reporter constructs containing the predicted miR-17-5p binding sites from both INTS6 and INTS6P1 to confirm direct interaction

Quantitative Correlation Analysis:

• Design a comprehensive tissue analysis protocol examining at least 30-40 paired tumor and normal samples to achieve statistical power .

• Develop a data integration pipeline that correlates:

  • INTS6 protein levels (detected via HRP-conjugated antibodies)

  • INTS6 mRNA levels (qRT-PCR)

  • INTS6P1 RNA levels (qRT-PCR)

  • miR-17-5p expression (qRT-PCR or in situ hybridization)

• Apply appropriate statistical methods including Spearman correlation for non-parametric data and multivariate analysis to account for confounding variables .

Functional Consequence Assessment:

• Design parallel experiments examining phenotypic outcomes following manipulation of each component:

This systematic approach will elucidate the regulatory circuit involving INTS6, INTS6P1, and miR-17-5p, providing insights into this novel mechanism of tumor suppression.

What optimization strategies yield the best results for detecting INTS6 in hepatocellular carcinoma samples?

Detecting INTS6 in hepatocellular carcinoma (HCC) samples requires comprehensive optimization of protocols specific to this challenging tissue type:

Immunohistochemistry Protocol Optimization:

• Tissue Processing Refinement:

  • Standardize fixation time to 24 hours in 10% neutral buffered formalin to prevent overfixation which can mask epitopes

  • Employ pressure cooker-based antigen retrieval with citrate buffer (pH 6.0) for 20 minutes at 120°C followed by 20 minutes cooling

  • Cut sections at 4μm thickness for optimal antibody penetration and consistent staining

• Blocking Optimization:

  • Implement dual blocking strategy: 3% H₂O₂ for 10 minutes to quench endogenous peroxidase activity followed by 5% normal goat serum for 30 minutes

  • Include avidin/biotin blocking if using amplification systems

  • HCC tissues often exhibit high background; consider additional blocking with 0.3% BSA in TBS-T

• Antibody Parameters:

  • Optimal dilution range of 1:200 (1μg/ml) for HRP-conjugated INTS6 antibodies has proven effective

  • Incubate at 4°C overnight in humidity chamber to maximize specific binding while minimizing background

  • Wash extensively (5 × 5 minutes) with TBS-T to remove unbound antibody

• Signal Development:

  • Optimize DAB development time (typically 5-10 minutes) with microscopic monitoring to prevent overdevelopment

  • Consider tyramide signal amplification for low abundance detection

  • Counterstain with hematoxylin for 30 seconds for optimal nuclear detail without obscuring DAB signal

Western Blot Optimization for HCC Samples:

• Extraction Protocol:

  • Use RIPA buffer supplemented with protease inhibitor cocktail, phosphatase inhibitors, and 1mM PMSF

  • Homogenize HCC tissues thoroughly using mechanical disruption at 4°C

  • Clarify lysates by centrifugation at 14,000g for 15 minutes at 4°C

• Electrophoresis Conditions:

  • Load 50μg protein per lane (higher than standard due to potentially low INTS6 expression in HCC)

  • Use gradient gels (4-12%) to optimize resolution of the ~90 kDa INTS6 protein

  • Include positive control (normal liver lysate) in each gel

• Transfer and Detection:

  • Employ wet transfer at 30V overnight at 4°C for efficient transfer of higher molecular weight proteins

  • Block membranes with 5% non-fat dry milk in TBST for 1 hour at room temperature

  • Incubate with HRP-conjugated INTS6 antibody at 1:1000 dilution for 2 hours at room temperature

  • Develop with enhanced chemiluminescence substrate and optimize exposure time based on signal intensity

Comparative Analysis Framework:

• Include multiple controls in each experimental run:

  • Normal liver tissue (positive control)

  • Known INTS6-negative sample (negative control)

  • Technical controls (no primary antibody)

• Standardize quantification methods using digital image analysis with calibrated exposure settings

• Consider multiplex staining to simultaneously assess INTS6 and relevant markers (e.g., Ki-67, α-fetoprotein)

These optimization strategies will maximize detection sensitivity and specificity for INTS6 in HCC samples, enabling reliable analysis of its expression patterns in this disease context.

How does the specificity and sensitivity of INTS6 Antibody, HRP conjugated compare across detection platforms?

The performance characteristics of INTS6 Antibody, HRP conjugated vary significantly across detection platforms, with important implications for experimental design and data interpretation:

Western Blotting Performance:

• Specificity: Western blotting provides high specificity (approximately 95-98%) due to molecular weight discrimination of the ~90 kDa INTS6 protein, effectively separating it from potential cross-reactive species .

• Sensitivity: Medium to high sensitivity with detection thresholds typically in the range of 0.1-1 ng of target protein when using enhanced chemiluminescence (ECL) detection systems.

• Quantitative Capacity: Semi-quantitative with a dynamic range of approximately 10-fold when properly calibrated with dilution series of control samples.

• Technical Limitations: Denatured protein conformation may affect epitope accessibility; membrane transfer efficiency can impact detection of larger proteins.

Immunohistochemistry Performance:

• Specificity: Moderate specificity (85-90%) dependent on antibody quality and epitope uniqueness; cross-reactivity is more challenging to control without the molecular weight separation .

• Sensitivity: Variable sensitivity heavily influenced by tissue fixation and antigen retrieval protocols; can detect INTS6 in FFPE tissues but may miss low-abundance expression.

• Quantitative Capacity: Limited to semi-quantitative scoring systems (0-3+ intensity, percentage positive cells); digital pathology analysis can improve quantitative assessment.

• Technical Advantages: Preserves tissue architecture and cellular context; allows spatial assessment of INTS6 expression within tissue microenvironment.

ELISA Performance:

• Specificity: High specificity (90-95%) in well-optimized systems using validated antibodies, though comprehensive validation is essential .

• Sensitivity: Generally high sensitivity with detection limits potentially reaching 10-50 pg/mL in sandwich ELISA formats.

• Quantitative Capacity: Superior quantitative performance with standard curves spanning 2-3 log ranges of concentration; coefficient of variation typically <10% for intra-assay measurements.

• Technical Advantages: High-throughput capacity; well-established quantitative framework; reduced technical complexity compared to microscopy-based methods.

Comparative Performance Analysis:

Validation Methodology:
When using HRP-conjugated INTS6 antibodies across multiple platforms, implement a cross-validation approach:

• Establish concordance between methods using the same sample set

• Deploy siRNA-mediated knockdown samples as specificity controls across all platforms

• Analyze correlation between protein detection (via antibody) and mRNA expression (via qRT-PCR)

• Consider the specific research question when selecting the optimal detection platform

This comprehensive understanding of platform-specific performance characteristics enables rational experimental design and appropriate interpretation of INTS6 detection data.

What systematic troubleshooting approach should be implemented when INTS6 detection yields inconsistent results?

When encountering inconsistent results with INTS6 Antibody, HRP conjugated, a systematic troubleshooting framework is essential for resolving technical issues:

Problem: Weak or Absent Signal

Sequential Diagnostic Approach:

• Antibody Integrity Assessment:

  • Evaluate storage history (-20°C or -80°C as recommended)

  • Check freeze-thaw cycles (should be <3)

  • Verify antibody expiration date and lot number

  • Resolution: Obtain new antibody or prepare fresh aliquots from master stock

• Epitope Accessibility Analysis:

  • For IHC: Test multiple antigen retrieval methods in parallel:

    • Heat-induced epitope retrieval with citrate buffer (pH 6.0)

    • EDTA buffer (pH 9.0)

    • Trypsin-based enzymatic retrieval

  • For Western blotting: Compare reducing vs. non-reducing conditions

  • Resolution: Optimize based on empirical testing of different conditions

• Expression Level Verification:

  • INTS6 is downregulated in approximately 70% of HCC tissues

  • Resolution: Include known positive controls (normal liver tissue)

  • Consider signal amplification methods (tyramide signal amplification for IHC; enhanced chemiluminescence for Western blotting)

  • Increase protein loading (up to 75μg for Western blotting)

• Protocol Optimization Matrix:

Problem: High Background or Non-specific Staining

Systematic Resolution Approach:

• Blocking Optimization:

  • Test blocking agent variants:

    • 5% BSA in TBS-T

    • 5% normal serum (species-matched to secondary antibody)

    • Commercial protein-free blockers

  • Extend blocking time from 30 minutes to 2 hours

  • Resolution: Select blocking conditions yielding lowest background with preserved specific signal

• Endogenous Enzyme Inactivation:

  • For HRP-conjugated antibodies, thorough quenching of endogenous peroxidase is critical

  • Resolution: Implement dual quenching approach:

    • 3% H₂O₂ for 10 minutes at room temperature

    • Commercially available peroxidase blocking reagents containing azide compounds

• Specificity Enhancement:

  • The 96% homology between INTS6 and its pseudogene INTS6P1 may complicate detection

  • Resolution: Validate specificity through parallel RNA interference experiments

  • Consider epitope-specific monoclonal antibodies if available

• Washing Protocol Refinement:

  • Insufficient washing is a common cause of high background

  • Resolution: Implement extended washing protocol (5 × 5 minutes with gentle agitation)

  • Use 0.1% Tween-20 in wash buffers to reduce non-specific interactions

Problem: Inconsistent Results Between Experiments

Standardization Implementation:

• Sample Preparation Standardization:

  • For tissues: Standardize fixation time (24h), processing protocol, and section thickness (4μm)

  • For cell lysates: Standardize cell confluence (70-80%), lysis buffer composition, and protein determination method

• Antibody Management System:

  • Implement antibody validation protocol for each new lot

  • Maintain master aliquots of validated antibody lots

  • Document lot numbers and correlation with experimental outcomes

• Environmental Variable Control:

  • Control temperature during critical steps (antibody incubation, development)

  • Standardize development times for chromogenic substrates

  • Implement consistent image acquisition parameters

Comprehensive Validation Strategy:

• Employ cell line panels with known INTS6 expression levels (e.g., normal hepatocytes vs. HCC cell lines)

• Include appropriate controls in every experiment:

  • Positive control (normal liver)

  • Negative control (primary antibody omission)

  • siRNA-treated samples as specificity controls

• Verify findings using orthogonal methods (e.g., IF vs. WB vs. IHC)

• Correlate protein detection with mRNA expression data

This systematic troubleshooting framework will help researchers achieve consistent and reliable results when working with INTS6 Antibody, HRP conjugated across various experimental settings.

What advanced multiplexed detection strategies can incorporate INTS6 Antibody, HRP conjugated to study tumor suppression pathways?

Multiplexed detection systems integrating INTS6 Antibody, HRP conjugated enable comprehensive analysis of tumor suppression pathways through advanced methodological approaches:

Sequential Multiplex Immunohistochemistry (mIHC):

• Chromogenic Multiplexing Protocol:

  • Apply HRP-conjugated INTS6 antibody as the initial layer

  • Develop with DAB substrate (brown)

  • Perform heat-mediated antibody stripping (pH 6.0 buffer at 95°C for 10 minutes)

  • Apply subsequent antibodies against pathway components

  • Use spectrally distinct chromogens for each marker:

    • Vector Red for second marker

    • Vector Blue for third marker

    • Vector Black for fourth marker

  • This approach enables visualization of up to 4 proteins on a single tissue section with standard brightfield microscopy

• Tyramide Signal Amplification (TSA) Multiplexing:

  • Optimize HRP-conjugated INTS6 antibody dilution for TSA system (typically 5-10× more dilute than standard protocols)

  • Apply antibody and develop with fluorophore-labeled tyramide (e.g., FITC-tyramide)

  • Perform heat inactivation of HRP enzyme (95°C for 10 minutes)

  • Repeat with additional antibodies using different fluorophore-labeled tyramides

  • This technique enables detection of 5-7 proteins on the same section with superior signal-to-noise ratios

Integrated Protein-RNA Detection Systems:

To investigate the regulatory network involving INTS6, INTS6P1, and miR-17-5p , implement combined detection approaches:

• Combined RNAscope®-Immunohistochemistry Protocol:

  • Perform RNA in situ hybridization for INTS6P1 and miR-17-5p using RNAscope® technology

  • Develop with appropriate chromogen or fluorophore

  • Follow with HRP-conjugated INTS6 antibody detection

  • This integrated approach enables simultaneous visualization of RNA and protein components of the regulatory network

• Spatial Transcriptomics Integration:

  • Perform IHC with HRP-conjugated INTS6 antibody on tissue sections

  • Image and digitally annotate IHC patterns

  • Process adjacent sections for spatial transcriptomics

  • Integrate protein and transcriptome data through computational alignment

  • This advanced approach reveals spatial relationships between INTS6 protein expression and broader transcriptional programs

Pathway-Focused Panel Design:

For studying INTS6's tumor suppressor function in HCC, a strategically designed multiplexed panel might include:

Technical Implementation Protocol:

• Panel Optimization Strategy:

  • Validate each antibody/probe individually before multiplexing

  • Determine optimal sequence (typically from lowest to highest abundance)

  • Validate stripping/inactivation efficiency between rounds

  • Implement appropriate controls for each marker

• Analysis Workflow:

  • Acquire whole slide images using multispectral imaging systems

  • Perform spectral unmixing to resolve overlapping signals

  • Apply tissue segmentation (tumor vs. stroma, nuclear vs. cytoplasmic)

  • Perform quantitative analysis of marker co-expression

  • Apply spatial statistics to analyze distribution patterns

• Validation Approach:

  • Compare multiplexed results with single-marker controls

  • Validate key findings with orthogonal methods (e.g., flow cytometry for cell lines)

  • Correlate protein expression patterns with functional outcomes

This comprehensive multiplexed detection strategy enables integrated analysis of the INTS6 tumor suppression pathway, providing mechanistic insights that would be impossible with conventional single-marker approaches.