rbm47 Antibody

What are the validated applications for RBM47 antibodies in experimental research?

RBM47 antibodies have been validated for several experimental techniques, with varying degrees of application success depending on the specific antibody clone and manufacturer. Based on the available research data, the following applications have been validated:

For optimal results, heat-mediated antigen retrieval with citrate buffer pH 6 is strongly recommended before commencing with IHC staining protocols .

What is the cellular and tissue distribution pattern of RBM47?

RBM47 shows distinct expression patterns across tissues and cellular compartments:

• Subcellular localization: Predominantly nuclear, consistent with its role in RNA processing

• Tissue distribution: Higher expression levels observed in liver and kidney

• Other positive tissues: Expression detected in uterus, thyroid gland, colon, prostate, and stomach tissues by immunohistochemical analysis

• Cancer cells: Variable expression across different cancer cell lines, with higher expression reported in Huh7 cells and lower expression in HCCLM3 cells

Single-cell sequencing analyses have revealed that RBM47 is enriched in CD163+ M2 macrophages in gliomas, suggesting a potential role in the tumor immune microenvironment .

How should sample preparation be optimized for RBM47 detection?

Optimal sample preparation for RBM47 detection varies by application:

For Western Blot:

• Effective lysis buffers include standard RIPA buffer with protease inhibitors

• Samples successfully tested include human fetal lung tissue, SW480 cells, A549 cells, and rat lung tissue

• Expected molecular weight: 64 kDa

For Immunohistochemistry:

• Heat-mediated antigen retrieval with citrate buffer pH 6 is critical for optimal staining

• Paraffin-embedded tissue sections at 4-6 μm thickness are recommended

• Overnight primary antibody incubation at 4°C typically produces better results than shorter incubations

For Immunofluorescence:

• Paraformaldehyde fixation (4%) followed by permeabilization with 0.1-0.2% Triton X-100

• Nuclear counterstaining with DAPI helps confirm the predominantly nuclear localization of RBM47

How can RBM47 antibodies be utilized to investigate its role in cancer progression?

RBM47 has shown both tumor suppressor and context-dependent functions across different cancer types. To investigate these roles:

• Combined IHC/IF with prognostic markers:

  • Co-staining RBM47 with established prognostic markers in gliomas (such as IDH status, 1p/19q co-deletion, or MGMT promoter methylation) to assess correlations with malignancy grades

  • RBM47 expression was found to positively correlate with WHO tumor grade in gliomas, with higher expression associated with poor prognosis

• Knockdown/overexpression studies with functional assays:

  • In hepatocellular carcinoma (HCC) studies, RBM47 knockdown and overexpression experiments revealed:

    • RBM47 overexpression increased apoptosis rates

    • RBM47 suppressed cell proliferation and colony formation

    • RBM47 inhibited migration and invasion capacities

• Relationship with molecular pathways:

  • RBM47 functions as a tumor suppressor in HCC by targeting UPF1, acting as both a DNA and RNA regulator

  • In gliomas, RBM47 expression was correlated with immune checkpoint molecules, particularly TIM-3 (correlation coefficient of 0.799 in TCGA dataset, p=1.5E-73)

What methodological approaches can elucidate RBM47's role in RNA regulation?

RBM47's diverse functions in RNA regulation can be investigated using several antibody-dependent techniques:

• RNA Immunoprecipitation (RIP) for identifying bound RNAs:

  • RBM47 antibodies can be used to immunoprecipitate RBM47-RNA complexes

  • Research has identified key mRNA targets including IFNAR1, with demonstrated enrichment in RBM47-Flag immunoprecipitation

  • The enriched RNA can be analyzed by qRT-PCR or RNA sequencing

• mRNA stability assays:

  • After RBM47 overexpression or knockdown, treat cells with RNA synthesis inhibitors like DRB or actinomycin D

  • Harvest cells at various time points (0, 15, 30, 45, 60 min) and measure target mRNA levels

  • RBM47 has been shown to delay the degradation of target mRNAs such as IFNAR1

• Co-immunoprecipitation for protein interaction studies:

  • RBM47 antibodies can help identify protein interaction partners

  • RBM47 has been shown to interact with APOBEC1 to form an mRNA editing complex involved in cytidine to uridine editing

How can RBM47 antibodies help investigate its role in immune responses?

RBM47 plays significant roles in immune regulation, particularly in interferon signaling. The following approaches using RBM47 antibodies can help elucidate these functions:

• Dual immunofluorescence staining with immune cell markers:

  • Co-staining of RBM47 with CD163 (M2 macrophage marker) revealed co-expression in glioma samples

  • This approach confirmed that RBM47 is enriched in M2 macrophages, suggesting a role in modulating the tumor immune microenvironment

• Analysis of interferon signaling pathway components:

  • Investigate the relationship between RBM47 and IFNAR1 using co-immunoprecipitation and western blot

  • RBM47 stabilizes IFNAR1 mRNA, leading to enhanced interferon downstream signaling

• Viral infection models:

  • RBM47 has demonstrated broad-spectrum antiviral activity against multiple viruses:

    • Dengue virus (DENV)

    • Zika virus (ZIKV)

    • Vesicular stomatitis virus (VSV)

    • Herpes simplex virus-1 (HSV-1)

  • Antibodies can be used to monitor RBM47 expression levels in response to viral infection or interferon treatment

What are the optimal conditions for studying RBM47's relationship with p53 signaling?

Recent research has identified connections between RBM47 and the p53-p21 axis:

• Co-immunoprecipitation approach:

  • Use RBM47 antibodies to pull down associated proteins

  • Western blot for p53 and p21 to detect potential interactions

  • RBM47 has been identified as an important regulator of basal and DNA damage-induced p53 and p21

• DNA damage response studies:

  • Treat cells with DNA-damaging agents (e.g., ionizing radiation)

  • Monitor RBM47, p53, and p21 protein levels by western blot

  • RBM47 has been shown to associate with ATM protein, a key sensor for DNA double-strand breaks

• Alternative splicing analysis:

  • Examine the impact of RBM47 on MDM4 mRNA alternative splicing

  • RBM47 binds to MDM4 mRNA and regulates its alternative splicing and expression, potentially affecting p53 activity

What experimental controls are essential when using RBM47 antibodies?

To ensure reliable results when using RBM47 antibodies, incorporate these critical controls:

• Antibody validation controls:

  • Positive controls: Tissues/cells known to express RBM47 (liver, kidney, or Huh7 cells)

  • Negative controls: RBM47 knockout or knockdown samples

  • Isotype controls: Particularly important for immunoprecipitation experiments

• RNA-binding specificity controls:

  • RNase treatment controls to confirm RNA-dependent interactions

  • Competition assays with purified RNA

  • Analysis of non-target RNAs as negative controls

• Functional validation controls:

  • Rescue experiments in knockdown/knockout models

  • Comparison of results using multiple antibody clones from different manufacturers

  • Use of tagged RBM47 constructs (e.g., RBM47-Flag) for validation of antibody specificity

How can non-specific binding be minimized when using RBM47 antibodies?

Several strategies can help minimize non-specific binding:

• Optimization of antibody dilution:

  • For western blot: Test dilutions in the 1:500-1:2000 range

  • For IHC: Starting dilution of 1:50-1:250 has been reported effective

• Blocking optimization:

  • 5% BSA in TBST often provides superior blocking compared to non-fat milk for RBM47 detection

  • Extended blocking times (1-2 hours at room temperature) may improve specificity

• Sample-specific considerations:

  • For tissues with high RBM47 expression (liver, kidney), more stringent washing conditions may be required

  • Reduction of primary antibody incubation time can sometimes improve signal-to-noise ratio

How should conflicting RBM47 antibody data be interpreted?

When faced with contradictory results using different RBM47 antibodies:

• Consider epitope differences:

  • Different RBM47 antibodies target distinct regions of the protein

  • Compare the specific epitopes targeted by each antibody (e.g., middle region, N-terminal, C-terminal)

• Evaluate protein isoforms:

  • Determine if conflicting results could be explained by detection of different RBM47 isoforms

  • Some antibodies may preferentially detect specific splice variants

• Cross-validation approaches:

  • Use multiple techniques (WB, IHC, IF) to confirm findings

  • Employ genetic approaches (siRNA, CRISPR) to validate antibody specificity

  • When possible, utilize orthogonal methods like mass spectrometry to confirm antibody findings

What are the best practices for long-term storage and handling of RBM47 antibodies?

To maintain antibody performance over time:

• Storage recommendations:

  • Store at -20°C as recommended by manufacturers

  • For antibodies provided in glycerol (typically 50%), aliquoting is often unnecessary for -20°C storage

  • For antibodies without glycerol, prepare small aliquots to avoid repeated freeze-thaw cycles

• Working solution preparation:

  • Prepare fresh working dilutions on the day of the experiment

  • If storage is necessary, keep at 4°C for no more than 1-2 weeks

  • Add preservatives like sodium azide (0.02%) for working solutions stored at 4°C

• Quality control monitoring:

  • Periodically test antibody performance with positive control samples

  • Monitor for changes in background or signal intensity over time

  • Document lot numbers and maintain consistency within experimental series