rbm47 Antibody, FITC conjugated

What is RBM47 and what cellular functions does it perform?

RBM47 is an RNA-binding protein containing three RNA recognition motifs (RRMs) that are essential for its biological function. Research demonstrates that RBM47 is an interferon-inducible protein that significantly enhances host interferon downstream signaling pathways . Unlike many immune modulators, RBM47 has no significant impact on interferon production itself, but rather amplifies the IFN-stimulated response element (ISRE) and enhances expression of interferon-stimulated genes (ISGs) .

Mechanistically, RBM47 functions by binding to the 3'UTR of target mRNAs such as IFNAR1, increasing their stability and retarding their degradation . This stabilization effect appears to be dependent on RBM47's RNA recognition domains, as mutants lacking these domains (ΔRRM) fail to enhance ISRE promoter activity or provide antiviral protection . Beyond its role in interferon signaling, RBM47 has been identified as a tumor suppressor in hepatocellular carcinoma and other cancers .

Why would researchers choose a FITC-conjugated RBM47 antibody over unconjugated versions?

FITC-conjugated antibodies offer several methodological advantages for RBM47 research:

• Direct detection without secondary antibodies, reducing experimental complexity and potential cross-reactivity issues

• Ideal for multi-parameter analysis when studying RBM47 alongside other proteins in the interferon pathway

• Enables direct visualization of subcellular localization through fluorescence microscopy

• Compatible with flow cytometric analysis for quantitative assessment of RBM47 expression levels

• Reduces background in co-localization studies with RNA using RNA-FISH techniques

The FITC fluorophore provides strong green fluorescence (excitation ~495nm, emission ~520nm) that works with standard laboratory equipment, making it suitable for laboratories investigating RBM47's role in antiviral immunity and cancer biology.

How does RBM47 contribute to antiviral immunity?

RBM47 demonstrates significant antiviral activity both in vitro and in vivo through several mechanisms:

• Enhancement of interferon signaling: RBM47 binds to and stabilizes IFNAR1 mRNA, increasing receptor expression and amplifying JAK-STAT pathway activation .

• Induction of ISGs: Experimental evidence shows RBM47 significantly increases the expression of interferon-stimulated genes (ISGs) that directly inhibit viral replication .

• Broad-spectrum antiviral effects: Studies demonstrate RBM47 effectively suppresses multiple RNA viruses, including dengue virus (DENV), Zika virus (ZIKV), and vesicular stomatitis virus (VSV) .

• In vivo protection: RBM47 heterozygous mice (RBM47+/-) exhibit increased susceptibility to viral infection, with higher viral loads and reduced ISG expression compared to wild-type mice .

This multi-level contribution to antiviral immunity makes RBM47 an important target for immunological research, particularly in studies examining host-pathogen interactions.

What are the optimal conditions for immunofluorescence staining using RBM47-FITC antibodies?

For optimal immunofluorescence results with FITC-conjugated RBM47 antibodies, researchers should follow this methodological protocol:

• Fixation: 4% paraformaldehyde for 15 minutes at room temperature (preserves both protein structure and fluorophore activity)

• Permeabilization: 0.2% Triton X-100 for 10 minutes (enables antibody access to intracellular RBM47)

• Blocking: 5% BSA in PBS for 1 hour (reduces non-specific binding)

• Antibody incubation: Dilute FITC-RBM47 antibody 1:100-1:200 in blocking buffer; incubate 2 hours at room temperature or overnight at 4°C

• Washing: 3-4 washes with PBS containing 0.1% Tween-20

• Counterstaining: DAPI for nuclear visualization (1:1000 dilution, 5 minutes)

• Mounting: Anti-fade mounting medium to reduce photobleaching

Throughout the procedure, samples should be protected from light to prevent FITC photobleaching. This protocol has been effective for visualizing both nuclear and cytoplasmic RBM47 distribution in the context of interferon signaling studies.

How should researchers design controls for RBM47-FITC antibody validation?

A comprehensive validation strategy for RBM47-FITC antibody should include:

Implementation of this control hierarchy ensures both technical reliability and biological relevance of the antibody staining results.

What experimental approaches can determine RBM47's RNA-binding specificity using FITC-conjugated antibodies?

To characterize RBM47's RNA-binding specificity, researchers should implement a multi-method approach:

• RNA Immunoprecipitation (RIP):

  • Crosslink protein-RNA complexes using formaldehyde or UV

  • Immunoprecipitate using FITC-conjugated RBM47 antibody

  • Extract bound RNAs for analysis by qRT-PCR or RNA sequencing

  • RIP assays have successfully identified IFNAR1 mRNA as a direct RBM47 target

• Electrophoretic Mobility Shift Assay (EMSA):

  • Generate biotin-labeled RNA probes from candidate target regions

  • Incubate with purified RBM47 protein

  • Observe mobility shift in non-denaturing polyacrylamide gel

  • EMSA confirmed direct interaction between RBM47 and IFNAR1 mRNA fragments

• Reporter Assays with RNA Fragments:

  • Clone putative binding sequences into luciferase reporter constructs

  • Test wild-type and mutant sequences for RBM47 responsiveness

  • Quantify changes in reporter expression relative to controls

  • This approach validated three functional binding sites in IFNAR1 3'UTR

• Cellular Validation:

  • Compare mRNA stability in wild-type versus RBM47 knockout cells

  • Perform actinomycin D chase experiments to measure mRNA half-life

  • Correlate with protein expression by Western blot

These complementary approaches provide robust evidence for direct RBM47-RNA interactions and their functional consequences.

How can researchers use RBM47-FITC antibodies to study protein localization changes during viral infection?

FITC-conjugated RBM47 antibodies enable dynamic tracking of RBM47 localization during viral infection through these methodological approaches:

• Time-Course Imaging:

  • Infect cells with virus (such as VSV or DENV)

  • Fix cells at defined intervals post-infection (0h, 2h, 6h, 12h, 24h)

  • Stain with RBM47-FITC antibody and counterstain nuclei

  • Image using confocal microscopy to track nuclear/cytoplasmic distribution

• Co-localization Analysis:

  • Perform dual staining with RBM47-FITC and markers of:

    • Processing bodies (P-bodies) using anti-DCP1a antibody

    • Stress granules using anti-G3BP1 antibody

    • Viral replication complexes using antibodies against viral proteins

  • Calculate Pearson's correlation coefficient to quantify co-localization

• Fractionation Studies:

  • Separate nuclear and cytoplasmic fractions at different infection timepoints

  • Analyze RBM47 distribution by flow cytometry using the FITC-conjugated antibody

  • Correlate with viral replication kinetics and interferon signaling activation

• Live-Cell Imaging (complementary approach):

  • Generate cells expressing RBM47-GFP fusion protein

  • Validate fusion protein functionality in RBM47 knockout background

  • Perform time-lapse imaging during infection to track real-time dynamics

This multi-faceted approach allows researchers to correlate RBM47 redistribution with key events in the viral life cycle and interferon response.

What strategies can resolve data inconsistencies when studying RBM47 in different cell types?

When faced with conflicting data on RBM47 function across cell types, researchers should implement these systematic troubleshooting approaches:

• Cell Type-Specific Expression Analysis:

  • Quantify baseline RBM47 expression in different cell types using flow cytometry with FITC-RBM47 antibody

  • Correlate with mRNA levels by qRT-PCR

  • Example: RBM47 showed variable expression patterns across 293T, HFF, HUVEC, and THP-1 cells

• Pathway Component Assessment:

  • Evaluate expression levels of key interferon pathway components

  • Measure IFNAR1/2, STAT1/2, and ISG expression by multiplexed analysis

  • Test responsiveness to exogenous interferon in each cell type

• Genetic Complementation:

  • Knock down endogenous RBM47 and rescue with standardized expression constructs

  • Compare functional outcomes using isogenic backgrounds

  • Test both full-length RBM47 and domain mutants (3RRM and ΔRRM variants)

• Target RNA Expression Analysis:

  • Compare abundance of RBM47 target RNAs (like IFNAR1) across cell types

  • Assess RNA stability and half-life in different cellular contexts

  • Examine alternative 3'UTR usage that might affect binding site availability

A comprehensive data table documenting RBM47 behavior across cell types can help identify patterns explaining apparent inconsistencies:

This systematic approach transforms apparent inconsistencies into valuable insights about context-dependent RBM47 activity.

How can RBM47-FITC antibodies be integrated into studies of interferon signaling during cancer progression?

RBM47-FITC antibodies can be powerful tools for investigating the intersection of interferon signaling and cancer biology through these methodological approaches:

• Tumor Microenvironment Analysis:

  • Perform immunofluorescence on tissue sections using RBM47-FITC antibody

  • Co-stain with markers of tumor cells, immune infiltrates, and IFN pathway activation

  • Quantify RBM47 expression in different cellular compartments

  • RBM47 has demonstrated tumor suppressor activity in hepatocellular carcinoma

• Correlation with Cancer Progression:

  • Stage-specific analysis of RBM47 expression in tumor samples

  • Correlate with patient outcomes and response to immunotherapy

  • Analyze relationship between RBM47 levels and key cancer hallmarks

• Mechanistic Studies:

  • Examine relationship between RBM47 and cancer-relevant targets:

    • UPF1 regulation in hepatocellular carcinoma

    • IFNAR1 stability in tumor cells versus surrounding tissue

    • ISG expression profiles in RBM47-high versus RBM47-low tumors

• Therapy Response Prediction:

  • Measure RBM47 expression before and after interferon-based therapies

  • Develop flow cytometry panels including RBM47-FITC to assess circulating tumor cells

  • Correlate RBM47 levels with response to immune checkpoint inhibitors

Evidence suggests RBM47's dual role as both tumor suppressor and interferon pathway enhancer makes it a valuable biomarker for understanding cancer immune evasion mechanisms and potential therapeutic vulnerabilities.

What optimizations are necessary for flow cytometric analysis using RBM47-FITC antibodies?

For optimal flow cytometric detection of RBM47 using FITC-conjugated antibodies, researchers should implement these technical optimizations:

• Sample Preparation Protocol:

  • Harvest cells in PBS with 2% FBS to maintain viability

  • Fix with 2-4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.1% saponin or 0.1% Triton X-100 (optimize for cell type)

  • Block with 5% normal serum for 30 minutes

  • Stain with titrated RBM47-FITC antibody (typically 0.5-2 μg per million cells)

• Instrument Settings Optimization:

  • Use 488nm laser for FITC excitation

  • Collect emission in 515-545nm range

  • Perform compensation if multiplexing with other fluorophores

  • Establish voltages using unstained and single-stained controls

• Controls Framework:

• Analysis Considerations:

  • Report data as median fluorescence intensity (MFI)

  • Calculate fold-change relative to appropriate controls

  • Consider histogram overlay for population comparisons

  • Use bivariate plots to correlate with other parameters (e.g., pSTAT1)

These optimizations ensure accurate quantification of RBM47 expression in complex cell populations and experimental conditions.

How should researchers troubleshoot weak signal when using RBM47-FITC antibodies?

When encountering weak signal with RBM47-FITC antibodies, implement this systematic troubleshooting approach:

• Expression-Level Assessment:

  • Confirm RBM47 expression in target cells by qRT-PCR

  • Consider stimulating cells with IFN-α (50-1000 U/ml for 6-24h) to upregulate RBM47

  • Use positive control cells with known high expression (e.g., transfected cells)

• Antibody Performance Optimization:

  • Titrate antibody concentration (test 2-5× recommended concentration)

  • Extend incubation time (overnight at 4°C rather than 1-2h)

  • Reduce washing stringency (decrease detergent concentration)

  • Test alternative fixation methods (compare PFA, methanol, acetone)

• Signal Enhancement Strategies:

  • Implement tyramide signal amplification (TSA) for immunofluorescence

  • Use anti-FITC antibody conjugated to brighter fluorophore (secondary enhancement)

  • Apply imaging settings optimizations (increased exposure, gain adjustment)

  • Consider antibody concentration using centrifugal filters

• Storage and Handling Evaluation:

  • Check antibody age and storage conditions

  • Minimize freeze-thaw cycles (aliquot upon receipt)

  • Protect from light throughout all procedures

  • Centrifuge antibody solution before use to remove aggregates

This methodical approach addresses both biological variables (expression levels) and technical factors affecting antibody performance.

What considerations apply when designing multiplexed assays incorporating RBM47-FITC antibodies?

For successful multiplexed assays incorporating RBM47-FITC antibodies, researchers should consider:

• Spectral Compatibility Planning:

  • FITC emission spectrum (peak ~520nm) overlaps with:

    • PE (partially)

    • GFP (significantly)

    • CFSE (significantly)

  • Recommended compatible fluorophores:

    • APC (660nm)

    • BV421 (421nm)

    • PE-Cy7 (785nm)

• Panel Design Strategy (Example for Interferon Pathway):

• Protocol Optimization:

  • Sequence staining steps carefully:

    • Surface markers first (if applicable)

    • Fixation and permeabilization

    • Intracellular targets including RBM47

  • Adjust compensation based on single-stained controls

  • Validate antibody combinations for potential interference

• Analysis Considerations:

  • Design bivariate plots to correlate RBM47 with pathway components

  • Use dimensionality reduction techniques (tSNE, UMAP) for high-parameter data

  • Implement Boolean gating to identify specific cell populations

• Biological Validation:

  • Confirm expected relationships (e.g., RBM47-high cells should show increased IFNAR1 and ISG expression)

  • Include appropriate stimulation controls (IFN-α treatment, viral infection)

  • Test in multiple cell types to confirm consistent patterns

This comprehensive approach ensures generation of meaningful multiplexed data that captures the biological complexity of RBM47's role in interferon signaling and antiviral immunity.