RNF6 Antibody, HRP conjugated

What is RNF6 and why is it significant in cancer research?

RNF6 is a zinc finger ubiquitin ligase that has been identified as a potential therapeutic target in several cancers. It plays crucial roles in cancer development through multiple mechanisms. In colorectal cancer, RNF6 overexpression accelerates carcinogenesis compared to wild-type controls in chemically induced models . RNF6 promotes colorectal cancer cell proliferation and invasion by transcriptionally upregulating splicing factor 3b subunit 2 (SF3B2) . In gastric cancer, RNF6 is upregulated in both primary tissues and cell lines, where it contributes to cancer cell growth by affecting the SHP-1/STAT3 signaling pathway . The molecular mechanism involves RNF6 regulating the stability of SHP-1 by inducing its polyubiquitination, which subsequently impacts STAT3 activation and downstream target genes like MCL1 and XIAP . Understanding these pathways has significant implications for developing targeted cancer therapies.

How does HRP conjugation enhance RNF6 antibody applications?

HRP (Horseradish peroxidase) conjugation to RNF6 antibodies provides several methodological advantages for research applications. The conjugation enables directional covalent bonding of HRP to the antibody, allowing for sensitive detection in various immunoassays without requiring secondary antibodies . This direct conjugation results in:

• Reduced background signal and improved signal-to-noise ratio

• Shortened experimental time by eliminating secondary antibody incubation steps

• Increased sensitivity for detecting low-abundance proteins like RNF6

• Greater consistency in experimental results due to standardized enzyme-to-antibody ratios

• Compatibility with multiple detection methods including colorimetric, chemiluminescent, and fluorescent substrates

The conjugation process maintains antibody functionality with high conjugation efficiency and 100% antibody recovery, even when performed at near-neutral pH with small quantities of antibody .

What are the primary experimental applications for RNF6 antibody, HRP conjugated?

RNF6 antibody, HRP conjugated, can be utilized in multiple experimental contexts:

These applications are particularly valuable for investigating RNF6's role in cancer development, as they allow researchers to examine RNF6 expression levels, protein-protein interactions, and DNA binding activities . For instance, ChIP-PCR assays with RNF6 antibodies have revealed that RNF6 binds to the SF3B2 promoter region at multiple sites, providing insight into its transcriptional regulatory functions .

How should RNF6 antibody, HRP conjugated be stored to maintain optimal activity?

For maximum stability and retention of activity, RNF6 antibody, HRP conjugated should be stored according to these research-validated guidelines:

• Store at -20°C in appropriate buffer conditions (typically containing 0.01M TBS at pH 7.4 with 1% BSA and 50% glycerol)

• Aliquot the antibody into multiple small volumes to avoid repeated freeze-thaw cycles, which can degrade both the antibody and HRP enzyme activity

• Include preservatives such as 0.03% Proclin300 to prevent microbial contamination during storage

• When in use, keep on ice or at 4°C and return to -20°C promptly

• Monitor storage buffer pH, as HRP activity is pH-sensitive and optimal at neutral pH

• Avoid exposure to strong oxidizing agents, reducing agents, and heavy metals, which can compromise HRP enzymatic activity

• Protect from prolonged light exposure, especially when working with photosensitive detection systems

Following these practices ensures consistent antibody performance across experiments and extends the usable life of the reagent.

How can RNF6 antibody, HRP conjugated be used to study ubiquitination dynamics?

RNF6 antibody, HRP conjugated, offers sophisticated approaches for investigating ubiquitination dynamics:

• Immunoprecipitation-Immunoblot (IP/IB) Assays: RNF6 antibody can be used to pull down RNF6 and its binding partners, followed by detection of ubiquitinated proteins. Studies have shown that RNF6 undergoes auto-ubiquitination, and treatments with proteasome inhibitors like MG132 and bortezomib increase RNF6 polyubiquitination in a concentration-dependent manner . The HRP conjugation enables direct detection of these complexes without secondary antibodies.

• K48 vs. K63 Linkage Analysis: Advanced research can distinguish between different polyubiquitin chain linkages. For example, P5091 (a USP7 inhibitor) markedly increases RNF6 at the K48-linked polyubiquitination form, which is associated with proteasomal degradation . HRP-conjugated antibodies allow for sensitive detection of these specific linkages.

• Ubiquitination Dynamics Assessment: When combined with cycloheximide (CHX) chase assays, RNF6 antibody, HRP conjugated, can determine the half-life and turnover rate of RNF6 and its targets. This approach has revealed that P5091 significantly reduces the half-life of RNF6 by increasing its turnover rate .

• Deubiquitinase (DUB) Interaction Studies: The antibody can identify interactions between RNF6 and deubiquitinating enzymes like USP7 and USP9x, which regulate RNF6 stability . These interactions can be visualized using precipitation methods followed by direct HRP detection.

What methodological approaches are recommended for analyzing RNF6's transcriptional regulation functions?

Recent research has established RNF6 as a transcriptional regulator, particularly in colorectal cancer. The following methodological approaches using RNF6 antibody, HRP conjugated, are recommended:

• Chromatin Immunoprecipitation (ChIP) Assays: ChIP-PCR and ChIP-qPCR have demonstrated that RNF6 binds to the SF3B2 promoter region at multiple sites, with a core binding motif sequence "TTTCCT" . For optimal results:

  • Use crosslinking conditions of 1% formaldehyde for 10 minutes at room temperature

  • Sonicate chromatin to fragments of 200-500 bp

  • Immunoprecipitate with 2-5 μg of RNF6 antibody per reaction

  • Include appropriate negative controls (IgG) and positive controls

• Electrophoretic Mobility Shift Assay (EMSA): EMSA can verify direct binding of RNF6 to DNA sequences:

  • Nuclear extracts expressing RNF6 from cancer cells have shown band shifts when incubated with SF3B2 promoter probes

  • Supershift assays using 0.1 or 0.4 μg of RNF6 antibody can confirm specificity

  • Include normal rabbit IgG as a control

  • Visualize using chemiluminescence detection methods

• Integrative Analysis: Combining ChIP-sequencing with RNA-sequencing has revealed downstream transcriptional targets of RNF6, including SF3B2 in colorectal cancer . This approach requires:

  • High-quality RNF6 antibody for ChIP-seq applications

  • Bioinformatic analysis to identify binding motifs and correlate with expression data

  • Functional validation of identified targets

• Luciferase Reporter Assays: To quantify the impact of RNF6 on target gene promoters:

  • Clone the promoter region containing RNF6 binding sites into a luciferase reporter

  • Compare reporter activity with RNF6 overexpression, knockdown, or mutation of binding sites

  • Normalize with appropriate controls to account for transfection efficiency

How can RNF6 antibody, HRP conjugated be utilized in studying cancer therapeutic responses?

RNF6 antibody, HRP conjugated, provides valuable tools for investigating therapeutic responses in cancer research:

• Monitoring RNF6 as a Biomarker: RNF6 expression levels correlate with cancer progression and treatment response. In gastric cancer, RNF6 is upregulated and its knockdown enhances the cytotoxicity of doxorubicin . The HRP-conjugated antibody enables precise quantification of RNF6 protein levels before and after treatment.

• Targeting RNF6-Dependent Pathways: Research has shown that:

  • In colorectal cancer, the RNF6-SF3B2 axis is a promising therapeutic target

  • Targeting this pathway with the SF3B2 inhibitor pladienolide B suppresses the growth of CRC cells with RNF6 overexpression both in vitro and in vivo

  • The combination of 5-fluorouracil (5-FU) plus pladienolide B demonstrates synergistic effects in CRC with high RNF6 expression, leading to tumor regression in xenograft models

• Mechanistic Studies of Drug Resistance: The antibody can be used to investigate:

  • How RNF6 expression affects response to standard chemotherapies

  • Whether RNF6 confers resistance through its effects on specific signaling pathways

  • The potential of RNF6 inhibition to overcome treatment resistance

• Evaluation of Targeted Therapies: For compounds targeting the ubiquitin-proteasome system:

  • USP7 inhibitors like P5091 accelerate RNF6 degradation by increasing K48-linked polyubiquitination

  • This approach can be monitored using the HRP-conjugated RNF6 antibody to detect protein degradation dynamics

• In vivo Efficacy Assessment: For preclinical animal studies:

  • Immunohistochemistry using RNF6 antibody, HRP conjugated, can evaluate target engagement in tumor tissues

  • Western blotting of tumor lysates can quantify RNF6 expression changes in response to therapy

What are the common challenges in Western blotting with RNF6 antibody, HRP conjugated, and how can they be addressed?

Researchers frequently encounter these challenges when using RNF6 antibody, HRP conjugated, for Western blotting:

When studying RNF6 specifically, it's important to note that its levels can be regulated by proteasomal degradation. Treatment with proteasome inhibitors like MG132 can significantly increase RNF6 protein levels in experimental systems . Additionally, RNF6 undergoes auto-ubiquitination, which can result in multiple bands on Western blots corresponding to different ubiquitinated forms .

How can experimental conditions be optimized for detecting RNF6-protein interactions?

Optimizing detection of RNF6-protein interactions requires careful consideration of methodological parameters:

• Immunoprecipitation Optimization:

  • Cell lysis conditions significantly impact protein complex preservation

  • Use NP-40 or CHAPS-based buffers with protease inhibitors for membrane-associated complexes

  • Include deubiquitinase inhibitors (e.g., N-Ethylmaleimide) to preserve ubiquitinated species

  • Research has shown that RNF6 interacts with both USP7 and USP9x proteins, which can be detected in RNF6 immunoprecipitates

• Crosslinking Approaches:

  • For transient interactions, consider chemical crosslinking with DSS or formaldehyde

  • Optimize crosslinker concentration and reaction time (typically 0.5-2 mM for 15-30 minutes)

  • Quench the reaction with glycine or Tris buffer before cell lysis

• Detection Strategies:

  • Direct detection with HRP-conjugated antibodies reduces background and improves sensitivity

  • For weak interactions, a sequential immunoprecipitation approach may be more effective

  • Consider native PAGE for intact complexes, particularly when studying E3 ligase activities

• Controls and Validation:

  • Include both positive controls (known interactors) and negative controls (IgG, irrelevant antibodies)

  • Validate interactions using reciprocal immunoprecipitation

  • For novel interactions, confirm with orthogonal methods (proximity ligation assay, FRET)

Studies have shown that the N-terminal TRAF or the C-terminal UBL domains are critical for USP7 to interact with RNF6, while RNF6 interacts with USP7 via its undefined domain (aa. 87–482) . This level of detail can guide the design of experiments to study specific protein-protein interactions.

What strategies can improve the sensitivity and specificity of RNF6 detection in clinical samples?

Clinical samples present unique challenges for RNF6 detection due to limited material, varying preservation methods, and complex tissue composition. These methodological approaches can enhance results:

• Signal Amplification Systems:

  • Tyramide signal amplification (TSA) can increase sensitivity by 10-100 fold

  • Employ polymer-based detection systems for improved signal-to-noise ratio

  • Consider quantum dot labeling for multiplexed detection and greater photostability

• Sample Preparation Optimization:

  • For FFPE tissues, optimize antigen retrieval methods (citrate pH 6.0 vs. EDTA pH 9.0)

  • Employ dual blocking strategy (protein block followed by peroxidase block)

  • Include tissue-matched controls for antibody validation

  • Consider automated staining platforms for consistency across samples

• Quantification Methods:

  • Implement digital image analysis for objective quantification

  • Use normalized H-score or quickscore methods accounting for both intensity and proportion

  • Compare results with orthogonal methods (qRT-PCR, mass spectrometry)

• Multiplex Detection Strategies:

  • Sequential immunohistochemistry for co-localization studies

  • Spectral unmixing for simultaneous detection of multiple markers

  • Combine with in situ hybridization to correlate protein and mRNA levels

Research has demonstrated that RNF6 is upregulated in both primary tissues and cell lines of gastric cancer , suggesting its potential utility as a biomarker. Optimized detection methods are crucial for accurately assessing its expression in diverse clinical samples.

How might RNF6 antibody, HRP conjugated be employed in studying cancer stem cells and therapeutic resistance?

Emerging research points to connections between ubiquitin ligases like RNF6 and cancer stem cell (CSC) biology, opening new investigative avenues:

• Cancer Stem Cell Identification and Isolation:

  • RNF6 antibody, HRP conjugated, can be used in flow cytometry-based approaches to identify potential CSC populations based on RNF6 expression

  • Magnetic-activated cell sorting (MACS) with RNF6 antibody could isolate CSC-enriched populations for functional studies

  • Correlation of RNF6 expression with established CSC markers could reveal new therapeutic targets

• Therapeutic Resistance Mechanisms:

  • RNF6 has been implicated in therapy resistance pathways, including STAT3 signaling in gastric cancer

  • The antibody can monitor changes in RNF6 expression before and after treatment to identify resistant populations

  • Combination therapies targeting RNF6-dependent pathways show promise in overcoming resistance, as demonstrated by the synergistic effects of 5-fluorouracil plus pladienolide B in colorectal cancer models

• Signaling Pathway Analysis:

  • RNF6 regulates multiple cancer-relevant pathways including:

    • SHP-1/STAT3 signaling in gastric cancer

    • SF3B2-dependent pathways in colorectal cancer

  • HRP-conjugated antibodies enable sensitive detection of these pathway components in limited sample material

• Preclinical Model Development:

  • Patient-derived xenografts with varying RNF6 expression levels can be characterized using the antibody

  • Organoid models can be evaluated for RNF6 expression patterns to predict drug responses

  • Genetic manipulation of RNF6 in these models can be verified and monitored using the antibody

What are the methodological considerations for investigating RNF6's role in alternative splicing regulation?

Recent research has identified connections between RNF6 and splicing factors, particularly SF3B2 in colorectal cancer . Investigating this emerging area requires specific methodological approaches:

• RNA-Seq Analysis for Alternative Splicing Events:

  • Compare splicing patterns in RNF6 overexpression, knockdown, and control conditions

  • Focus on exon skipping, intron retention, alternative 5'/3' splice sites, and mutually exclusive exons

  • Validate with RT-PCR using exon-spanning primers for selected targets

• Splicing Reporter Assays:

  • Employ minigene constructs containing alternatively spliced regions

  • Evaluate how RNF6 expression levels affect splicing decisions

  • Use the HRP-conjugated antibody to confirm RNF6 expression levels in these experiments

• RNA-Protein Interaction Analysis:

  • RNA immunoprecipitation (RIP) using RNF6 antibody can identify directly bound RNA targets

  • Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) provides nucleotide-resolution binding maps

  • Functional validation through site-directed mutagenesis of binding sites

• Proteomic Analysis of Spliceosome Components:

  • Identify RNF6-dependent changes in spliceosome composition

  • Monitor post-translational modifications of splicing factors

  • Investigate how RNF6-mediated ubiquitination affects splicing factor activity

• Therapeutic Targeting Strategies:

  • SF3B2 inhibitor pladienolide B has shown efficacy against RNF6-overexpressing colorectal cancer

  • Develop combination approaches targeting both RNF6 and downstream splicing factors

  • Use the HRP-conjugated antibody to monitor target engagement in preclinical models

How can chromatin remodeling effects of RNF6 be investigated using the HRP-conjugated antibody?

RNF6's role as a transcriptional regulator suggests potential involvement in chromatin remodeling processes. These methodological approaches can shed light on this emerging area:

• Chromatin Immunoprecipitation Followed by Sequencing (ChIP-seq):

  • Generate genome-wide binding profiles for RNF6 using the HRP-conjugated antibody

  • Correlate binding sites with histone modification patterns and chromatin accessibility

  • Analyze binding motifs to identify co-factors and regulatory elements

• Chromatin Interaction Analysis:

  • Chromosome conformation capture (3C) and its derivatives can identify long-range interactions influenced by RNF6

  • Proximity ligation assay can visualize RNF6 associations with specific chromatin regions

  • HiChIP approaches can map RNF6-associated chromatin interactions genome-wide

• Epigenetic Modification Analysis:

  • Investigate how RNF6 binding correlates with specific histone modifications

  • Determine whether RNF6 recruits histone-modifying enzymes to target loci

  • Monitor changes in DNA methylation patterns at RNF6 binding sites

• Nucleosome Positioning and Occupancy:

  • ATAC-seq can reveal changes in chromatin accessibility upon RNF6 manipulation

  • MNase-seq can identify alterations in nucleosome positioning and occupancy

  • Combined with RNF6 ChIP-seq data, these approaches can reveal mechanistic insights

Research has demonstrated that RNF6 binds to the SF3B2 promoter region at multiple sites , but broader effects on chromatin structure and function remain to be fully characterized. The HRP-conjugated antibody provides a sensitive tool for investigating these emerging roles.