rbck1 Antibody

What are the primary applications of RBCK1 antibodies in research?

RBCK1 antibodies are utilized in multiple experimental techniques, including western blotting (WB), immunoprecipitation (IP), immunofluorescence (IF), immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA). These applications enable researchers to detect, localize, and quantify RBCK1 protein in various sample types. When selecting an RBCK1 antibody, consider its validated reactivity with your species of interest (commonly human, mouse, or rat) and its performance in your specific application .

What is the molecular weight of RBCK1 for western blot detection?

RBCK1 typically appears at approximately 57-58 kDa in western blot applications. The calculated molecular weight is 58 kDa, while the observed molecular weight is generally around 57 kDa . When performing western blot with RBCK1 antibodies, it's advisable to include positive controls, such as placenta tissue lysates, which have been validated for RBCK1 detection .

What cellular compartments should RBCK1 be detected in?

RBCK1 shuttles between the nucleus and cytoplasm, possessing both nuclear export and localization signals within its amino acid sequence . When performing immunofluorescence or immunohistochemistry, expect to observe RBCK1 staining in both compartments. Co-localization studies have shown that RBCK1 interacts with various proteins, including RNF31, as demonstrated through immunofluorescence analysis . For optimal subcellular localization experiments, use confocal microscopy and appropriate counterstains for nuclear and cytoplasmic markers.

How should I design knockdown experiments to study RBCK1 function?

For effective RBCK1 knockdown experiments:

• Design multiple siRNAs targeting different regions of RBCK1 mRNA (at least 6 pairs recommended based on published protocols)

• Validate knockdown efficiency using both western blotting and real-time PCR

• Select the two most effective siRNAs for functional studies

• Include appropriate controls (siControl/scrambled siRNA)

• Verify knockdown persistence throughout your experimental timeframe

Studies in ER-positive breast cancer cells (T47D and MCF-7) and hepatocellular carcinoma cells (PLC/PRF/5 and huh-7) have successfully employed this approach, demonstrating significant phenotypic changes following RBCK1 silencing .

What cell lines are most appropriate for studying RBCK1 function in cancer?

Based on published research, the following cell lines have been validated for RBCK1 functional studies:

When selecting cell lines, consider RBCK1 expression levels, as U87MG and A172 cell lines have been reported to express relatively high levels of RBCK1 protein .

What are the recommended controls for RBCK1 antibody validation?

For rigorous RBCK1 antibody validation:

• Positive tissue controls: Human placenta tissue has been validated for RBCK1 detection in WB, IHC, and IP applications

• Knockdown/knockout controls: Use RBCK1 siRNA or CRISPR/Cas9-mediated knockout cells alongside wild-type cells

• Peptide competition: Pre-incubate antibody with immunizing peptide to confirm specificity

• Multiple antibody comparison: Validate findings with at least two different RBCK1 antibodies from different sources or clones (e.g., clone H-1 sc-393754 and clone E-2 sc-365523)

• Cross-reactivity assessment: Test antibody reactivity in species-specific positive and negative controls

These validation steps are crucial for ensuring specificity and reproducibility in RBCK1-focused research.

How does RBCK1 regulate the HIF1α pathway in cancer cells?

RBCK1 regulates HIF1α through a post-translational mechanism involving the ubiquitin-proteasome pathway:

• RBCK1 interacts directly with HIF1α protein

• This interaction indirectly inhibits the polyubiquitination of HIF1α specifically at the K48 ubiquitination site

• By preventing ubiquitination, RBCK1 enhances HIF1α protein stability and extends its half-life

• The RBR (RING-B-Box-RING) domain of RBCK1, particularly the RING domain, plays a crucial role in this process

• Increased HIF1α stability leads to enhanced expression of HIF1α target genes (e.g., VEGFA, SLC2A1, BNIP3)

This mechanism has been comprehensively characterized in ER-positive breast cancer cell lines through ubiquitination IP experiments, protein stability assays, and rescue experiments . To investigate this pathway, researchers should employ CHX chase assays, proteasome inhibitors (MG132), and ubiquitination immunoprecipitation techniques alongside site-directed mutagenesis of the RBCK1 RING domain (e.g., Flag-RBCK1 C406A plasmid) .

What techniques can be used to study RBCK1 protein-protein interactions?

For investigating RBCK1 protein interactions:

• Co-immunoprecipitation (Co-IP): Demonstrated effective for detecting RBCK1 interactions with RNF31 and HIF1α

• Immunofluorescence co-localization: Used to visualize RBCK1 co-localization with interaction partners like RNF31

• Proximity ligation assay (PLA): Provides high-sensitivity detection of protein-protein interactions in situ

• GST pulldown assays: Useful for mapping specific interaction domains

• FRET or BRET analysis: For studying dynamic interactions in living cells

• Yeast two-hybrid screening: For identifying novel interacting partners

When performing Co-IP for RBCK1, use antibodies targeting distinct epitopes for immunoprecipitation and detection to minimize background. Cell lysis conditions should be optimized to preserve native protein complexes while ensuring efficient extraction.

How should I design functional rescue experiments to confirm RBCK1 mechanism?

Based on successful rescue experiments in published RBCK1 research:

• Silence RBCK1 using validated siRNAs in your cell model

• Verify knockdown efficiency at both mRNA and protein levels

• Simultaneously overexpress the hypothesized downstream effector (e.g., HIF1α)

• For HIF1α rescue experiments, use a mutant plasmid with enhanced stability to overcome the short half-life of native HIF1α

• Assess rescue through multiple functional assays (e.g., migration, invasion, colony formation)

• Include appropriate controls: siControl + empty vector, siRBCK1 + empty vector, siControl + effector overexpression

This approach has been successfully employed to demonstrate that HIF1α overexpression rescues the inhibitory effects of RBCK1 knockdown on cell migration, wound healing, and colony formation in MCF-7 cells .

How is RBCK1 expression correlated with clinical outcomes in cancer patients?

RBCK1 expression correlates with clinical outcomes across several cancer types:

For clinical correlative studies, researchers should employ multiple approaches including TCGA database analysis, tissue microarray IHC, and correlation with established clinical parameters. When performing IHC scoring of RBCK1, establish clear scoring criteria based on staining intensity and percentage of positive cells, and employ at least two independent pathologists for evaluation .

What is the relationship between RBCK1 expression and response to cancer therapies?

RBCK1 expression has been associated with differential therapeutic responses:

• Anti-angiogenic therapy: High RBCK1 expression correlates with increased sensitivity to anti-angiogenic agents (Axitinib, Masitinib, Pazopanib, Sorafenib) in glioma, potentially due to RBCK1's role in promoting angiogenesis through upregulation of endothelial cell-specific genes

• Immunotherapy: Elevated RBCK1 expression is associated with higher TIDE scores and increased T cell exclusion in glioma, predicting poorer responses to immune checkpoint blockade therapy

To study RBCK1's impact on therapy response, researchers should:

• Perform drug sensitivity assays using multiple anti-angiogenic compounds

• Analyze correlations between RBCK1 expression and endothelial cell-specific markers (CLEC14A, PECAM1, CDH5, CLDN5)

• Assess T cell infiltration and exclusion through multiplex immunofluorescence or RNA-seq-based immune cell deconvolution algorithms

How should patient tissues be processed for optimal RBCK1 detection?

For optimal RBCK1 detection in patient tissues:

• Fixation: 10% neutral-buffered formalin for 24-48 hours

• Antigen retrieval: Two options have been validated:

  • TE buffer (pH 9.0) - preferred method

  • Citrate buffer (pH 6.0) - alternative approach

• Antibody dilution: 1:50-1:500 for IHC applications (optimize for specific antibody and tissue type)

• Detection system: Use polymer-based detection systems with appropriate positive controls

• Storage: For long-term storage, store antibody at -20°C; for short-term (up to 6 months), 4°C storage is sufficient

For clinical research applications, validation across multiple tissue types and comparison with normal adjacent tissues is essential for accurate interpretation of RBCK1 expression patterns.

How can RNA-seq data be leveraged to understand RBCK1-regulated pathways?

RNA-seq analysis has provided valuable insights into RBCK1-regulated pathways:

• Experimental design: Compare control cells to RBCK1-knockdown cells (minimum triplicate samples)

• Conditions: For hypoxia-related studies, subject cells to 12h hypoxia before RNA extraction

• Analysis approaches:

  • Gene Set Enrichment Analysis (GSEA) - identified HIF1α signaling pathway correlation

  • KEGG pathway analysis - revealed RBCK1's involvement in multiple signaling pathways

  • Volcano plotting - identified classical target genes affected by RBCK1 (e.g., VEGFA, SLC2A1, BNIP3)

RNA-seq data from MCF-7 breast cancer cells (GSE196274) demonstrated that RBCK1 knockdown inhibits the HIF1α signaling pathway . When analyzing RNA-seq data, utilize appropriate normalization methods and false discovery rate correction for differential expression analysis, followed by pathway enrichment tools like GSEA, DAVID, or STRING.

What are the emerging techniques for studying RBCK1's E3 ligase activity?

Advanced techniques for investigating RBCK1's E3 ligase function include:

• In vitro ubiquitination assays: Reconstitution of the ubiquitination cascade using purified components

• Ubiquitin linkage-specific antibodies: For distinguishing K48 vs. other ubiquitin chain types

• UbiCREST assay: Utilizing linkage-specific deubiquitinating enzymes to determine chain types

• UbiSite and diGly proteomics: For global identification of RBCK1 substrates

• TUBE (Tandem Ubiquitin Binding Entities): For enrichment of ubiquitinated proteins

• Structure-function studies of RBCK1's RBR domain: Particularly the RING domain which has been demonstrated crucial for its function

Research has demonstrated that the UbcH7(C86K)-Ub conjugate binds to the RBCK1 RBR-helix in the presence of the allosteric activator M1 di-Ub . For functional studies, the RING domain mutation (C406A) has been validated as disrupting RBCK1's ability to regulate ubiquitination .

What are the methodological approaches for studying RBCK1 in the tumor microenvironment?

For investigating RBCK1's role in the tumor microenvironment:

• Tumor-conditioned medium (TCM) experiments:

  • Collect medium from RBCK1-knockdown or overexpressing tumor cells

  • Apply to endothelial cells (HUVECs) to assess functional effects

  • Measure migration, apoptosis, and tube formation

• Immune infiltration analysis:

  • Correlate RBCK1 expression with immune cell markers in RNA-seq or microarray data

  • Utilize multiplex immunofluorescence to assess spatial relationships

  • Analyze immune checkpoint gene correlations (LAG3, PD-1, CTLA4, PD-L1)

• Tumor microenvironment models:

  • Orthotopic tumor models with RBCK1 manipulation

  • Co-culture systems with immune cells and stromal components

  • Patient-derived organoids maintaining TME components

RBCK1 has been associated with tumor immune evasion mechanisms, T cell exclusion, and angiogenesis in glioma . For comprehensive TME studies, combine computational approaches (TIDE algorithm, immune cell deconvolution) with experimental validation (FACS analysis of tumor-infiltrating lymphocytes, multiplex IHC).

These methodological approaches provide a framework for investigating RBCK1's complex roles in cancer biology and potential therapeutic implications.