RBCK1 Antibody, HRP conjugated

What experimental applications are suitable for RBCK1 Antibody, HRP conjugated?

RBCK1 Antibody with HRP conjugation is validated for multiple research applications including western blotting (WB), immunoprecipitation (IP), immunofluorescence (IF), and enzyme-linked immunosorbent assay (ELISA) . The HRP conjugation provides direct detection capability without requiring secondary antibodies, offering advantages in reducing background signal and simplifying experimental workflows.

For researchers beginning work with this antibody, it is recommended to first validate specificity in your specific experimental system. The experimental approach should include:

• Initial testing using recommended dilutions for your application (e.g., 1:1000-1:5000 for WB)

• Including both positive controls (tissues/cells known to express RBCK1) and negative controls

• For IP applications, using 0.5-4.0 μg antibody for 1.0-3.0 mg of total protein lysate

• For IHC applications, applying antigen retrieval with TE buffer pH 9.0 or alternatively citrate buffer pH 6.0

What is the species reactivity and specificity of RBCK1 antibody?

The most commonly used RBCK1 antibodies demonstrate reactivity with mouse, rat, and human samples . When selecting an RBCK1 antibody for your research, it is important to confirm species reactivity in the specific catalog information. For example, antibody 26367-1-AP specifically shows confirmed reactivity with human samples .

Cross-reactivity testing should be performed when working with novel species or sample types. The antibody recognizes RBCK1 protein's structural epitopes, which may be conserved across species but should be validated experimentally. When performing western blotting, the expected molecular weight for human RBCK1 should be verified against the manufacturer's datasheet.

What are the recommended dilutions and sample preparation protocols?

Optimal dilutions vary by application and should be determined empirically for each experimental system:

For sample preparation:

• For protein extraction, use RIPA buffer supplemented with protease inhibitors

• For IHC applications, antigen retrieval with TE buffer pH 9.0 is recommended, or alternatively citrate buffer pH 6.0

• For IF studies, 4% paraformaldehyde fixation followed by 0.1% Triton X-100 permeabilization is typically effective

Each new batch of antibody should be titered to determine optimal concentration for the specific application and experimental system being used .

How can RBCK1 Antibody, HRP conjugated be used to investigate the NF-κB signaling pathway?

RBCK1 has been demonstrated to participate in canonical NF-κB activation as part of the linear ubiquitin chain assembly complex (LUBAC) with SHARPIN and HOIP . When investigating this pathway, researchers should consider several methodological approaches:

• Co-immunoprecipitation studies: Use RBCK1 antibody to pull down associated proteins and probe for NF-κB pathway components.

  • Following IP, analyze for presence of HOIP and SHARPIN to confirm LUBAC formation

  • Evaluate phosphorylation status of IκBα and p65 as indicators of pathway activation

• Expression correlation analysis: In glioma research, RBCK1 knockdown decreased expression of p-IκBα and p-p65, suggesting direct pathway involvement .

• Signaling pathway analysis: Monitor multiple points in the pathway simultaneously:

  • IκBα phosphorylation and degradation

  • p65 phosphorylation and nuclear translocation

  • Downstream gene expression changes

When designing these experiments, it is essential to include appropriate controls and time course analyses, as NF-κB signaling is dynamic and context-dependent.

What methodological considerations are critical when studying RBCK1's role in cancer immunology?

RBCK1 has demonstrated significant associations with tumor immunity across multiple cancer types, particularly in glioma . When investigating these relationships, researchers should:

• Consider multiple immune markers simultaneously: RBCK1 expression correlates with various immune checkpoint molecules including LAG3, and to a lesser extent, PD-1 (PDCD1), CTLA4, and PD-L1 (CD274) .

• Analyze genomic correlation data: RBCK1 expression shows varying correlations with:

  • Tumor mutational burden (TMB)

  • Microsatellite instability (MSI)

  • Neoantigen presence

  • Stemness indices

• Incorporate immune cell infiltration analyses: When studying tumor samples, quantify:

  • T cell populations (particularly cytotoxic T cells)

  • MDSC (myeloid-derived suppressor cells) presence

  • T cell exclusion programs

• Evaluate therapy response correlations: Research has shown that glioma patients with higher RBCK1 expression:

  • Exhibit higher TIDE scores (indicating poorer responses to immunotherapy)

  • Show increased response to anti-angiogenic therapies

  • Display significantly higher scores in T cell exclusion programs

These experimental approaches should incorporate both in vitro and in vivo models when possible, as RBCK1's immunomodulatory effects may be dependent on the complete tumor microenvironment.

How should researchers interpret contradictory RBCK1 expression data across different experimental systems?

When encountering contradictory results regarding RBCK1 expression or function, researchers should systematically evaluate several factors:

• Antibody validation: Confirm that the RBCK1 antibody has been properly validated for your specific application:

  • Verify using alternative antibody clones

  • Perform knockdown/knockout validation experiments

  • Use recombinant protein positive controls

• Cell-type specific effects: RBCK1 functions differently across cell types and cancer types:

  • In glioma, RBCK1 is overexpressed in tumor tissues compared to para-cancerous brain tissues

  • Expression is significantly upregulated in Grade IV glioma compared to lower grades

  • Protein levels vary between cell lines (e.g., higher in U87MG and A172 cell lines)

• Subcellular localization context: RBCK1 shuttles between cytoplasm and nucleus, possessing both nuclear export and localization signals . Analysis should include:

  • Subcellular fractionation studies

  • Co-localization with known interaction partners

  • Nuclear vs. cytoplasmic expression ratios

• Post-translational modifications: Consider that RBCK1 function is regulated by modifications that may not be detected by all antibody clones.

What approaches can be used to investigate RBCK1's impact on angiogenesis and vascular remodeling?

Research has established RBCK1's involvement in angiogenesis, particularly in glioma . When studying this relationship, consider these methodological approaches:

• Gene expression correlation analysis:

  • Analyze correlation between RBCK1 and endothelial cell-specific genes (CLEC14A, PECAM1, CDH5, CLDN5)

  • Examine VEGFA expression changes following RBCK1 knockdown or overexpression

• Functional assays with endothelial cells:

  • Collect tumor-conditioned medium (TCM) from RBCK1-knockdown or overexpression cells

  • Assess migration capacity of HUVECs exposed to this medium

  • Measure apoptosis rates in HUVECs following exposure

  • Evaluate tube formation capacity

• HIF-1α pathway analysis:

  • Utilize luciferase reporter gene assays with VEGFA promoter regions

  • Results indicate RBCK1 upregulation enhances HIF-1α luciferase reporter activities

• Drug sensitivity testing:

  • Evaluate anti-angiogenic agent efficacy in relation to RBCK1 expression

  • Data shows that four anti-angiogenic drugs (Axitinib, Masitinib, Pazopanib, and Sorafenib) had lower IC50 values in RBCK1-high groups

How can researchers effectively compare RBCK1 antibody performance across different experimental protocols?

When comparing experimental results using RBCK1 antibodies across different protocols or laboratories, researchers should implement standardized comparison methods:

• Antibody validation across platforms:

  • Perform parallel experiments with the same samples using different detection methods

  • Verify epitope specificity using peptide competition assays

  • When possible, use knockout/knockdown controls across all platforms

• Standard curve calibration:

  • For quantitative applications, establish standard curves using recombinant RBCK1 protein

  • Normalize results to housekeeping proteins specific to each application

  • Include common positive control samples across experiments

• Cross-platform validation:

  • When moving between techniques (e.g., from WB to IHC), validate findings using complementary methods

  • Confirm protein-level findings with mRNA expression data when appropriate

  • Document all protocol variables that might influence antibody performance:

    • Fixation methods and duration

    • Antigen retrieval conditions

    • Blocking reagents used

    • Incubation times and temperatures

• Reporting standards:

  • Document complete antibody information (clone, catalog number, lot)

  • Report exact dilutions used rather than ranges

  • Specify detection systems and imaging parameters

These approaches help ensure reproducibility and valid comparisons across different experimental conditions, reducing the likelihood of contradictory results stemming from methodological differences rather than biological variables.

What experimental approaches should be used to study RBCK1's E3 ubiquitin ligase activity?

RBCK1 functions as an E3 ubiquitin-protein ligase, facilitating ubiquitin transfer from E2 ubiquitin-conjugating enzymes to target substrates . When investigating this activity:

• In vitro ubiquitination assays:

  • Combine purified components: E1, E2 (particularly UBE2L3), RBCK1, ubiquitin, ATP

  • Include appropriate controls (reactions lacking individual components)

  • Analyze ubiquitination patterns using western blotting

• Substrate identification:

  • Perform IP-mass spectrometry following RBCK1 pull-down

  • Validate potential substrates through in vitro and in vivo ubiquitination assays

  • Mutational analysis of predicted ubiquitination sites on target proteins

• Chain linkage analysis:

  • Use linkage-specific antibodies to determine types of ubiquitin chains formed

  • Research indicates that the UbcH7(C86K)-Ub conjugate binds to the RBCK1 RBR-helix in the presence of the allosteric activator M1 di-Ub

  • Study both K48 and K63 linkages, as well as linear ubiquitination

• Structure-function relationship:

  • Utilize RBCK1 domain mutants to map regions required for E3 activity

  • Analyze the RING-B-Box-Coiled-Coil (RBCC) structure's contribution to enzyme function

  • Investigate homodimerization, which enhances transcriptional and DNA-binding activities

These approaches provide comprehensive analysis of RBCK1's enzymatic function in both isolated biochemical systems and cellular contexts.

How can researchers effectively investigate RBCK1's role in immunotherapy resistance?

Evidence suggests RBCK1 may contribute to immunotherapy resistance, particularly in glioma . To investigate this relationship:

These approaches provide a comprehensive framework for understanding how RBCK1 may contribute to immunotherapy resistance mechanisms.

What are the most common technical issues when using RBCK1 Antibody, HRP conjugated, and how can they be resolved?

Researchers frequently encounter several technical challenges when working with RBCK1 antibodies. Here are solutions to common issues:

• High background in western blots:

  • Increase blocking time and concentration (try 5% BSA instead of milk for phospho-specific detection)

  • Reduce primary antibody concentration (test serial dilutions)

  • Increase washing frequency and duration

  • For HRP-conjugated antibodies specifically, ensure no cross-reactivity with blocking agents

• Weak or no signal detection:

  • Verify protein expression in your sample (use positive control tissues)

  • Optimize protein loading (increase concentration)

  • For membrane proteins, ensure proper extraction techniques

  • Consider extended exposure times for detection

  • For IHC applications, optimize antigen retrieval (test both citrate buffer pH 6.0 and TE buffer pH 9.0)

• Multiple bands or unexpected band sizes:

  • Verify if RBCK1 has known isoforms or post-translational modifications in your sample type

  • Include knockout/knockdown controls to confirm specificity

  • Optimize gel percentage and running conditions

  • Consider using gradient gels for better resolution

• Variable results between experiments:

  • Standardize all protocol steps including sample preparation, incubation times, and temperatures

  • Prepare larger volumes of antibody dilutions to use across multiple experiments

  • Document lot numbers and prepare for lot-to-lot variations

  • Include internal controls in each experiment for normalization

Systematic optimization of these parameters will help ensure consistent and reliable results when working with RBCK1 antibodies.

How should researchers design knockdown/knockout validation experiments for RBCK1 antibody specificity?

Proper validation of RBCK1 antibody specificity using genetic approaches should follow these methodological guidelines:

• siRNA-based knockdown:

  • Design at least 2-3 different siRNA sequences targeting different regions of RBCK1 mRNA

  • Include non-targeting control siRNA

  • Verify knockdown efficiency at both mRNA level (qPCR) and protein level (western blot)

  • Optimal knockdown should show >70% reduction in expression

• shRNA-based stable knockdown:

  • Implement inducible shRNA systems when studying long-term effects

  • Verify knockdown persistence over experimental timeframe

  • Examples show successful RBCK1 knockdown in U118MG and A712 cells

• CRISPR/Cas9 knockout validation:

  • Design guide RNAs with minimal off-target effects

  • Generate single-cell clones and verify complete knockout

  • Sequence the targeted region to confirm gene editing

  • Validate complete protein loss using the RBCK1 antibody

• Rescue experiments:

  • Reintroduce wild-type RBCK1 in knockout cells

  • Use expression vectors resistant to the knockdown approach

  • Confirm restoration of RBCK1-dependent phenotypes and antibody signal

• Controls and documentation:

  • Always include wild-type cells as positive controls

  • Document all validation steps thoroughly

  • Consider the effects of RBCK1 loss on cell viability, as complete knockout may be lethal in some cell types

These validation approaches establish antibody specificity while also providing valuable experimental tools for functional RBCK1 studies.

What emerging research areas could benefit from RBCK1 antibody applications?

Several cutting-edge research areas show promise for RBCK1 antibody applications:

• Precision oncology approaches:

  • Development of RBCK1 expression as a biomarker for anti-angiogenic therapy response

  • Research indicates glioma patients with elevated RBCK1 levels display increased responsiveness to anti-angiogenic therapies like regorafenib

  • Potential for companion diagnostic development using RBCK1 IHC

• Combinatorial therapy investigations:

  • Study of RBCK1 inhibition in combination with immune checkpoint blockade

  • Evidence suggests that targeting RBCK1 could potentially overcome resistance to immunotherapy

  • Analysis of synergistic effects with anti-angiogenic agents

• Single-cell analysis applications:

  • Application of RBCK1 antibodies in single-cell protein profiling

  • Investigation of heterogeneous expression patterns within tumor microenvironments

  • Correlation with other immune and angiogenic markers at single-cell resolution

• Structural biology and drug discovery:

  • Use of antibodies as tools to understand RBCK1 protein conformation

  • Development of conformation-specific antibodies to detect active vs. inactive states

  • Application in screening potential small molecule inhibitors of RBCK1

• Developmental biology:

  • Investigation of RBCK1's role in normal tissue development and homeostasis

  • Comparison with pathological functions in cancer and inflammatory conditions

  • Potential applications in regenerative medicine research

These emerging areas represent high-impact opportunities for application of RBCK1 antibodies in advancing scientific understanding and therapeutic development.

How can RBCK1 antibodies contribute to developing novel therapeutic approaches?

RBCK1 antibodies can facilitate therapeutic development through several research avenues:

• Target validation studies:

  • Use antibodies to confirm RBCK1 expression in patient-derived samples

  • Correlate expression with clinical outcomes and therapy responses

  • Data from glioma patients shows RBCK1 shapes an immunosuppressive tumor microenvironment

• Therapeutic antibody development:

  • Use existing research antibodies to identify accessible epitopes

  • Develop functional antibodies that could inhibit RBCK1's E3 ligase activity

  • Explore antibody-drug conjugate approaches targeting RBCK1-expressing cells

• Combination therapy rationale:

  • Investigate RBCK1 expression changes following standard therapies

  • Evidence suggests combining RBCK1-targeted therapy with anti-angiogenic agents may be beneficial

  • The IC50 values of four anti-angiogenic drugs (Axitinib, Masitinib, Pazopanib, and Sorafenib) were lower in RBCK1-high groups

• Biomarker development:

  • Standardize RBCK1 IHC protocols for potential diagnostic applications

  • Create scoring systems correlating with therapy response prediction

  • Develop companion diagnostics for anti-angiogenic therapy selection