rbm47 Antibody, Biotin conjugated

What is RBM47 and what are its primary biological functions?

RBM47 is a multifunctional RNA-binding protein that contains three RNA recognition motifs (RRMs) and primarily binds to mRNAs, most prominently in introns and 3'UTRs . Its main functions include:

• Regulating mRNA stability and abundance for a subset of target mRNAs

• Altering splicing patterns of target transcripts

• Acting as an RNA chaperone that can affect various post-transcriptional processes

• Enhancing interferon (IFN) downstream signaling by stabilizing IFNAR1 mRNA

• Modulating the Wnt signaling pathway through stabilization of DKK1 mRNA in some contexts

The functional importance of RBM47 varies across different tissue types and disease states, with significant implications in cancer biology and immune responses.

What experimental applications is the biotin-conjugated RBM47 antibody suitable for?

The biotin-conjugated RBM47 antibody is suitable for multiple research applications, particularly those benefiting from avidin-biotin detection systems. Based on RBM47 antibody properties, suitable applications include:

• Western blotting (WB) at dilutions of 1:500-1:2000

• Immunoprecipitation (IP) for protein-protein or protein-RNA interaction studies

• Immunohistochemistry (IHC) for tissue localization studies

• RNA immunoprecipitation (RIP) to identify RNA targets

• High-throughput immunoprecipitation and sequencing (HITS-CLIP) analysis for transcriptome-wide binding studies

• Flow cytometry for cellular detection and quantification

• ELISA for protein quantification

For optimal results, researchers should validate the biotin-conjugated antibody in their specific experimental system and perform appropriate titration to determine optimal concentrations.

How should researchers optimize protocols when using biotin-conjugated RBM47 antibody for Western blotting?

When optimizing Western blot protocols with biotin-conjugated RBM47 antibody, researchers should consider these methodological approaches:

• Sample preparation:

  • For cell lines like A549 or tissues like rat lung that show positive detection , use RIPA buffer with protease inhibitors

  • Heat samples at 95°C for 5 minutes in reducing sample buffer

• Electrophoresis parameters:

  • Use 10-12% SDS-PAGE gels since RBM47's molecular weight is approximately 64 kDa

  • Load adequate protein amount (20-50 μg total protein)

• Blocking optimization:

  • Critical consideration: Use biotin-free blocking reagents to prevent high background

  • Recommended: 5% BSA or specialized blocking reagents designed for biotin-streptavidin systems

• Antibody dilution:

  • Start with manufacturer-recommended dilution of 1:500-1:2000

  • Optimize through titration experiments

• Detection system:

  • Use streptavidin-HRP or streptavidin-fluorophore conjugates

  • Implement stringent washing steps to minimize background

• Controls:

  • Include RBM47 knockdown/knockout samples as negative controls

  • Use human, mouse, or rat samples as the antibody shows reactivity with these species

The expected band size for RBM47 is 64 kDa , which should be confirmed during validation experiments.

What are the recommended storage conditions for maintaining biotin-conjugated RBM47 antibody activity?

To maintain optimal activity of biotin-conjugated RBM47 antibody:

• Storage temperature: Store at -20°C as the primary storage condition

• Buffer composition: The antibody is typically preserved in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Aliquoting recommendations: Aliquoting is not required for -20°C storage of small volumes (20 μl) , but larger volumes should be aliquoted to prevent repeated freeze-thaw cycles

• Stability period: The antibody remains stable for one year after shipment when stored properly

• Handling precautions:

  • Avoid repeated freeze-thaw cycles

  • Keep on ice during experiments

  • Protect from prolonged light exposure (particularly important for biotin conjugates)

  • Centrifuge briefly before opening to collect solution at the bottom of the tube

These storage recommendations ensure maximum retention of antibody specificity and binding capacity for research applications.

How can researchers use RBM47 antibody to investigate its role in cancer progression, particularly in renal cell carcinoma?

Researchers investigating RBM47's role in renal cell carcinoma (RCC) can employ biotin-conjugated RBM47 antibodies through these advanced methodological approaches:

• Expression analysis in clinical specimens:

  • Perform tissue microarray analysis with RBM47 antibody to correlate expression with patient outcomes

  • Conduct quantitative immunohistochemistry to evaluate expression patterns across RCC subtypes and stages

  • Studies show RBM47 is downregulated in RCC tissues and negatively correlates with patient prognosis

• Chromatin regulation studies:

  • Investigate RBM47 expression regulation through ChIP assays

  • RBM47 expression in RCC is regulated by CBP/P300-mediated H3K27ac , which can be examined using sequential ChIP with H3K27ac and relevant transcription factor antibodies

• Signaling pathway analysis:

  • Examine RBM47's role in p53 signaling pathway through co-immunoprecipitation experiments

  • RBM47 interferes with HOXB-AS1/p53 protein interactions, promoting p53 nuclear entry and pathway activation

• Therapeutic response investigation:

  • Develop immunoblotting protocols to examine RBM47 expression in patient-derived xenografts before and after treatment

  • RBM47 demonstrates synergistic anticancer effects with sunitinib in RCC models

• RNA-protein interaction mapping:

  • Perform RIP-seq using the antibody to identify RBM47-binding RNAs in RCC cells

  • Design validation experiments for key targets like HOXB-AS1

A multi-modal approach combining these techniques would provide comprehensive insights into how RBM47 restrains RCC progression through its RNA-binding functions.

What troubleshooting approaches should be employed when conflicting RBM47 expression data is observed between antibody-based methods and RNA sequencing?

When researchers encounter discrepancies between RBM47 protein detection (using antibodies) and mRNA expression (from RNA-seq), systematic troubleshooting is essential:

• Biological explanations for discrepancies:

  • Post-transcriptional regulation: RBM47 itself regulates RNA stability , potentially creating feedback loops

  • Protein stability variations: Different cellular contexts may alter RBM47 protein half-life

  • Context-dependent expression: RBM47 shows tissue-specific expression patterns and disease-specific alterations

• Technical validation approaches:

  • Antibody validation:

    • Perform knockdown/knockout experiments to confirm antibody specificity

    • Test multiple RBM47 antibody clones targeting different epitopes

    • Use recombinant RBM47 protein as a positive control

  • RNA measurement validation:

    • Confirm RNA-seq findings with qRT-PCR using multiple primer sets

    • Examine splice variants that might affect antibody recognition sites

  • Cross-platform validation:

    • Correlate protein levels with mRNA expression in a panel of cell lines

    • Use nascent RNA labeling to distinguish transcriptional vs. post-transcriptional effects

• Resolution strategies for ongoing research:

  • Create data integration tables comparing protein and RNA measurements across samples

  • Develop mathematical models accounting for RNA stability and protein turnover

  • Consider cell type heterogeneity, particularly in complex tissues like gliomas where RBM47 shows enrichment in specific cell populations like CD163+ macrophages

This systematic approach helps distinguish biological phenomena from technical artifacts when evaluating RBM47 expression patterns.

How can biotin-conjugated RBM47 antibody be utilized in RNA immunoprecipitation sequencing (RIP-seq) to identify RNA targets across different disease models?

Implementing RIP-seq using biotin-conjugated RBM47 antibody requires careful methodological consideration:

• Protocol optimization for different disease models:

  • Cancer cell lines: For renal carcinoma (786-O, 769-P) or breast cancer metastasis models

  • Immune cells: When studying interferon responses where RBM47 enhances ISRE activity

  • Neural tissue: For investigating RBM47's role in gliomas

• Crosslinking considerations:

  • UV crosslinking: Optimal for direct RNA-protein interactions

  • Formaldehyde crosslinking: Suitable for capturing indirect interactions in RNP complexes

  • No crosslinking: For high-affinity interactions but may lose transient binding events

• Biotin-streptavidin capture optimization:

  • Use magnetic streptavidin beads for efficient capture

  • Implement stringent wash conditions to reduce non-specific binding

  • Include appropriate RNase inhibitors throughout the procedure

  • Consider on-bead library preparation to reduce sample loss

• Controls and normalization strategies:

  • Input normalization: Essential for distinguishing enrichment from abundance

  • IgG control: Critical for identifying non-specific binding

  • Knockdown validation: Compare RIP-seq results from cells with and without RBM47 knockdown

  • Spike-in controls: Consider using synthetic RNA for technical normalization

• Data analysis pipelines:

  • Motif discovery: Analyze for enriched sequence elements, particularly in 3'UTRs and introns where RBM47 shows prominent binding

  • Pathway analysis: Examine target RNAs for functional enrichment

  • Integration with stability data: Correlate binding with changes in RNA half-life

  • Cross-reference with published RBM47 targets like HOXB-AS1 and IFNAR1

This comprehensive approach allows researchers to generate high-confidence maps of RBM47-RNA interactions relevant to specific disease contexts.

What experimental designs are recommended to investigate the differential roles of RBM47 across cancer types, given its apparently contradictory functions?

RBM47 shows context-dependent functions across cancer types: tumor-suppressive in breast cancer and renal cell carcinoma , but potentially oncogenic in gliomas . To investigate these seemingly contradictory roles:

• Comparative molecular profiling experimental design:

  Analysis Type	Breast Cancer	Renal Cell Carcinoma	Glioma
  RBM47 expression	Downregulated	Downregulated	Upregulated
  Prognostic impact	Good when expressed	Good when expressed	Poor when expressed
  RNA targets	DKK1, others	HOXB-AS1	Immune-related transcripts
  Signaling impact	Suppresses metastasis	Activates p53 pathway	Affects immune environment

• Cross-cancer functional genomics approach:

  • Perform parallel RBM47 overexpression and knockdown in multiple cancer models

  • Use isogenic cell line panels representing each cancer type

  • Implement rescue experiments with cancer-specific RNA targets

  • Conduct xenograft studies comparing metastatic potential across models

• Target identification across cancer types:

  • Perform comparative RIP-seq in breast, renal, and glioma cell lines

  • Identify common and cancer-specific RNA targets

  • Validate with reporter assays for key targets

  • Create Venn diagrams of binding targets across cancer types

• Context-dependent protein interaction studies:

  • Conduct BioID or proximity labeling studies to identify cancer-specific protein partners

  • Perform co-immunoprecipitation with biotin-conjugated RBM47 antibody

  • Map interactome differences that might explain functional divergence

  • Examine post-translational modifications unique to each cancer context

• Microenvironment influence assessment:

  • Co-culture experiments with stromal or immune cells

  • Particularly relevant for gliomas where RBM47 is enriched in CD163+ macrophages

  • Compare RBM47 function under hypoxic vs. normoxic conditions

  • Test inflammatory cytokine effects on RBM47 function

This systematic approach would help reconcile the divergent roles of RBM47 across cancer types and potentially identify context-specific therapeutic strategies.

How can researchers effectively use biotin-conjugated RBM47 antibody to investigate its role in interferon signaling and antiviral responses?

For investigating RBM47's role in interferon signaling and antiviral immunity, researchers can employ biotin-conjugated RBM47 antibody in these methodologically advanced approaches:

• Interferon-stimulated response element (ISRE) regulation studies:

  • Use chromatin immunoprecipitation (ChIP) with biotin-conjugated RBM47 antibody to examine association with ISRE-containing promoters

  • Combine with luciferase reporter assays to quantify RBM47's impact on ISRE activity

  • Compare results in wildtype vs. RBM47+/- mouse models or knockdown cellular systems

• mRNA stability assessment protocols:

  • Design actinomycin D chase experiments to measure IFNAR1 mRNA stability

  • Use the antibody in RNA immunoprecipitation to confirm direct binding to IFNAR1 mRNA 3'UTR

  • Implement pulse-chase labeling with 4sU to measure newly synthesized vs. degraded RNA populations

• Protein domain functionality investigation:

  • Design co-immunoprecipitation experiments comparing wildtype RBM47 with domain mutants:

    • Full-length RBM47

    • 3RRM variant (containing RNA recognition motifs)

    • ΔRRM variant (lacking RNA recognition motifs)

  • Correlate binding patterns with functional readouts like VSV-GFP replication inhibition

• Viral infection experimental design:

  • Use the antibody to track RBM47 localization during viral infection

  • Implement time-course studies examining RBM47-RNA interactions during infection

  • Compare effectiveness against different virus families since RBM47 shows broad-spectrum antiviral activity

  • Quantify viruses like DENV, ZIKV, and VSV-GFP using appropriate assays

• Signaling cascade analysis:

  • Investigate JAK-STAT pathway amplification through phospho-specific antibody detection

  • Create an experimental matrix comparing IFN treatment conditions with/without RBM47 modulation

  • Measure ISG expression profiles in different cellular contexts

This comprehensive approach would elucidate how RBM47 enhances antiviral immunity through stabilizing IFNAR1 mRNA and amplifying downstream interferon signaling.

What reference standards and controls should be incorporated when validating a new lot of biotin-conjugated RBM47 antibody?

Comprehensive validation of new biotin-conjugated RBM47 antibody lots requires structured quality control protocols:

• Positive control selection matrix:

  Cell/Tissue Type	Validation Status	Expected Signal	Notes
  A549 cells	Confirmed positive	Strong	Human lung adenocarcinoma cell line
  Rat lung tissue	Confirmed positive	Moderate	Primary tissue positive control
  786-O, 769-P cells	Experimental models	Variable	Renal cell carcinoma lines
  Recombinant RBM47	Absolute standard	Strong	Use for specificity testing

• Negative control strategy:

  • RBM47 knockout/knockdown samples created using validated shRNAs (sh47-1, sh47-2, sh47-3)

  • Cell lines with naturally low RBM47 expression

  • Isotype control antibodies for non-specific binding assessment

  • Peptide competition assays to verify epitope specificity

• Cross-reactivity assessment:

  • Test against human, mouse, and rat samples as the antibody shows reactivity with these species

  • Perform Western blot analysis under reducing and non-reducing conditions

  • Check for signal at expected 64 kDa molecular weight

• Biotin conjugation quality metrics:

  • Determine degree of labeling (DOL) through spectrophotometric analysis

  • Measure free biotin percentage to ensure proper conjugation

  • Confirm streptavidin binding efficiency

  • Compare signal-to-noise ratio with unconjugated antibody version

• Lot-to-lot comparison protocol:

  • Side-by-side testing with previous lot as reference standard

  • Quantitative assessment of staining intensity and pattern consistency

  • Documentation of binding affinity through dose-response curves

  • Analysis of background levels across applications

This systematic validation approach ensures experimental reproducibility and confidence in research findings generated using the antibody.

What emerging applications and methodologies might benefit from biotin-conjugated RBM47 antibodies in studying RNA-binding protein networks?

Emerging technologies and methodological innovations offer new frontiers for biotin-conjugated RBM47 antibody applications:

• Spatial transcriptomics integration:

  • Combine immunofluorescence using biotin-conjugated RBM47 antibody with in situ sequencing

  • Map the spatial distribution of RBM47 protein in relation to its target RNAs

  • Particularly valuable in heterogeneous tissues like gliomas where RBM47 shows enrichment in specific cell populations

• Single-cell multi-omics approaches:

  • Develop protocols for simultaneous protein and RNA detection at single-cell resolution

  • Correlate RBM47 protein levels with transcript profiles

  • Investigate cell-type specific functions, especially in immune cell subsets given RBM47's role in interferon signaling

• RNA modification analysis pipelines:

  • Investigate whether RBM47 preferentially binds modified RNAs

  • Combine with epitranscriptomic sequencing methods

  • Assess competitive binding with other RNA-binding proteins at modified sites

• Liquid-liquid phase separation (LLPS) studies:

  • Examine RBM47's potential role in forming ribonucleoprotein granules

  • Use biotin-conjugated antibody for live-cell imaging of potential LLPS events

  • Combine with optogenetic tools to manipulate RBM47 assemblies

• Therapeutic monitoring applications:

  • Develop companion diagnostic approaches for cancer therapies

  • Particularly relevant for sunitinib treatment in renal cell carcinoma where RBM47 shows synergistic anticancer effects

  • Create multiplexed assays to monitor RBM47 and key pathway components

• CRISPR screening validation:

  • Use the antibody to validate hits from CRISPR screens targeting RBM47 regulatory factors

  • Implement cellular phenotyping based on RBM47 expression levels

  • Create reporter systems for high-throughput drug screening targeting RBM47 biology

These emerging applications highlight the continued value of high-quality RBM47 antibodies for advancing our understanding of RNA regulation in health and disease contexts.