RBM5 Antibody

What is RBM5 and why is it an important research target?

RBM5 (RNA binding motif protein 5) is a 815 amino acid protein containing two RRM (RNA recognition motif) domains and two zinc finger domains (ZnF) . It belongs to the RBM5/RBM10 family and functions as a component of the spliceosome A complex . RBM5 regulates alternative splicing of numerous mRNAs and may modulate splice site pairing after recruitment of the U1 and U2 snRNPs . Importantly, RBM5 plays dual roles in regulating apoptosis through alternative splicing of genes like FAS and CASP2/caspase-2 .

RBM5 has gained significant research interest because:

• It shows the highest expression in leukemia across all cancer types

• It is significantly overexpressed in acute myeloid leukemia (AML)

• It is down-regulated in colorectal cancer (CRC) tissues and cells

• It is involved in regulating alternative splicing in Huntington's disease

• It may be a key step in small cell lung cancer (SCLC) development

How do I select the most appropriate RBM5 antibody for my research?

When selecting an RBM5 antibody, consider these essential factors:

Advanced researchers should review publications that have used the antibody of interest and examine any validation data including knockout/knockdown controls.

What are the recommended dilutions for RBM5 antibodies in different applications?

Based on commercially available RBM5 antibodies, here are the recommended dilutions:

Note: Always perform antibody titration in your specific experimental system to determine optimal conditions.

What is the optimal protocol for RBM5 Western blotting?

For successful RBM5 Western blotting:

• Sample Preparation:

  • Use NETN lysis buffer for optimal extraction

  • Include protease inhibitors to prevent degradation

  • Load 15-50 μg of total protein per lane (based on published protocols)

• Gel Electrophoresis:

  • Use 8-10% SDS-PAGE gels (RBM5 is ~92 kDa calculated, but observed at 110-115 kDa)

  • Include molecular weight markers covering 75-120 kDa range

• Transfer and Blocking:

  • Transfer to PVDF membrane at 100V for 60-90 minutes

  • Block with 5% non-fat milk in TBST for 1 hour at room temperature

• Antibody Incubation:

  • Primary antibody: Dilute according to manufacturer's recommendation (typically 1:1000-1:6000)

  • Incubate overnight at 4°C

  • Secondary antibody: Anti-rabbit HRP (most RBM5 antibodies are rabbit polyclonal)

  • Recommended controls: RBM5 knockdown or knockout samples as negative controls

• Detection:

  • Use chemiluminescence detection with 30 seconds to 3 minutes exposure time

  • Expected band: 92 kDa (calculated) but often observed at 110-115 kDa

How should I optimize RBM5 immunohistochemistry for different tissue types?

Based on published protocols and commercial antibody recommendations:

• Tissue Preparation:

  • Fixation: 10% neutral buffered formalin

  • Section thickness: 4-5 μm paraffin sections

• Antigen Retrieval:

  • Primary method: TE buffer pH 9.0 heat-induced epitope retrieval

  • Alternative method: Citrate buffer pH 6.0

  • Heat treatment: 95-98°C for 15-20 minutes

• Tissue-Specific Considerations:

  • Human stomach, lung, and rat testis tissues have been validated for positive staining

  • Human brain tissue has shown positive signals with certain antibodies

• Staining Protocol:

  • Blocking: 3% H₂O₂ (10 min) followed by 5-10% normal serum

  • Primary antibody: Dilute 1:50-1:1000 based on the specific antibody

  • Incubation: Overnight at 4°C or 60 minutes at room temperature

  • Detection: HRP-polymer or ABC method with DAB substrate

• Controls:

  • Positive tissue controls: Lung tissue (high RBM5 expression)

  • Negative controls: Primary antibody omission or isotype controls

What experimental design should I use to study RBM5's role in cancer?

Based on published studies of RBM5 in cancer:

• Expression Analysis:

  • Compare RBM5 expression between tumor and matched normal tissues

  • Research shows RBM5 is highly overexpressed in AML compared to matched normal tissue

  • In contrast, RBM5 is downregulated in colorectal cancer tissues and cells

  • Use both mRNA (qRT-PCR) and protein (Western blot, IHC) analyses

• Functional Studies:

  • Loss-of-function approaches:

    • CRISPR/Cas9-mediated knockout: Validated in MOLM13, THP1, and OCIAML2 cells

    • RNA interference (shRNA): Two independent shRNAs (RBM5: sh#1 and sh#2) were effective

  • Gain-of-function approaches:

    • Overexpression using pcDNA3.1-RBM5 plasmid (validated in SW480 and HCT116 cells)

    • Use sgRNA-resistant RBM5 cDNA for rescue experiments

• Phenotypic Assays:

  • Cell proliferation: Cell viability assays, EDU incorporation

  • Cell differentiation: CD11b and CD14 expression markers

  • Apoptosis: Flow cytometry with Annexin V/PI staining

  • Cell migration and invasion: Transwell assays

  • Glycolysis: Measure glucose consumption, lactate production, ATP production

• In Vivo Models:

  • Xenograft studies: Inject luciferase-GFP-labeled cells (e.g., MOLM13) with RBM5 manipulation

  • Analyze tumor volume, tumor weight, and metastasis

  • Assess splenomegaly and leukemia cell infiltration in bone marrow, spleen, and peripheral blood

How can I study RBM5's RNA binding specificity and splicing regulation?

To investigate RBM5's RNA interactions and splicing functions:

• RNA Immunoprecipitation (RIP):

  • Use validated antibodies for RIP at 2 μg/ml concentration

  • Follow with qRT-PCR for specific targets or RNA-Seq for genome-wide analysis

  • RBM5 has been shown to bind to PTEN mRNA to stabilize its expression in colorectal cancer

• pCLAP (Peptide Cross-Linking and Affinity Purification):

  • This technique identified three RNA-binding peptides from RBM5 in mouse brain tissue

  • These peptides mapped to the two RRM domains

  • One peptide (spanning amino acids 104-115) showed differential binding in Huntington's disease model

• Alternative Splicing Analysis:

  • RBM5 regulates alternative splicing of FAS and CASP2/caspase-2

  • For FAS, RBM5 promotes exclusion of exon 6, producing a soluble form that inhibits apoptosis

  • For CASP2, RBM5 promotes exclusion of exon 9, producing a catalytically active form that induces apoptosis

  • Use RT-PCR with exon-specific primers or RNA-seq with splicing analysis tools

• Structure-Function Analysis:

  • RBM5 contains two RRM domains that are non-canonical (lacking consensus aromatic residue in RNP2)

  • Both RRM domains are essential for RNA binding - deletion of these domains abolishes RNA-binding ability

How do I interpret differential RBM5 expression across cancer types?

RBM5 shows tissue-specific and cancer-specific expression patterns that require careful interpretation:

• Leukemia/AML:

  • RBM5 shows highest expression in leukemia across all cancer types (TCGA data)

  • Significantly higher in AML compared to matched normal tissue

  • Particularly elevated in CEBPA mutated, NPM1-mutated and KMT2A-r subtypes

  • Functions as a pro-survival factor in AML cells - knockdown impairs growth and induces differentiation

• Colorectal Cancer (CRC):

  • RBM5 is significantly downregulated in CRC tissues and cells compared to normal controls

  • Functions as a tumor suppressor - overexpression inhibits proliferation, migration, invasion, and glycolysis

  • Mechanism involves binding to PTEN mRNA, stabilizing its expression, and inhibiting the PI3K/AKT pathway

• Lung Cancer:

  • The earliest and most frequent genetic alterations in lung cancers involve the region to which RBM5 maps

  • Decreased RBM5 expression may be a key step in small cell lung cancer (SCLC) development

  • RBM5 and RBM10 have distinct and sometimes opposing roles in lung cancer

To properly interpret these differences:

• Consider tissue-specific functions and protein interaction networks

• Evaluate RBM5 in the context of molecular subtypes of each cancer

• Analyze both mRNA and protein expression (they may not always correlate)

• Consider the impact of mutations in related pathways

What is the relationship between RBM5 and other RBM family proteins?

RBM5 has complex interactions with other RBM family members, particularly RBM10:

• RBM5 and RBM10 Relationship:

  • Both proteins have tumor-suppressor properties in various cancer cell lines

  • RBM5 expression can influence RBM10 expression

  • RBM5 post-transcriptionally regulates RBM10 expression via direct interaction with specific RBM10 splice variants

  • Despite similarities, they can have opposite functions in certain contexts:

    • In RBM5-null SCLC cell line (GLC20), RBM10 promoted cell proliferation and transformation-associated processes

• Structural Similarities and Differences:

  • Both contain multiple RNA recognition domains

  • RBM5 contains two RRM domains, two zinc finger domains (ZnF1 and ZnF2), an OCRE domain, and a G-patch domain

  • The RRM domains of RBM5 are non-canonical, lacking the consensus aromatic residue in RNP2

• Functional Cross-Regulation:

  • RBM5 and RBM10 cross-regulate each other

  • In RBM5-null tumors, RBM10 expression may contribute to tumor growth and metastasis

  • Measurement of both RBM10 and RBM5 expression in clinical samples may hold prognostic value

How can I troubleshoot inconsistent RBM5 Western blot results?

When encountering problems with RBM5 Western blotting:

Important note: RBM5 has been documented to appear at a higher molecular weight (110-115 kDa) than its calculated size (92 kDa) in Western blots , which may reflect post-translational modifications.

How should I interpret contradictory findings about RBM5's role in different cancers?

RBM5 exhibits context-dependent functions across different cancers:

• Establish Tissue Context:

  • RBM5 functions as an oncogene in AML (supports leukemia cell survival)

  • RBM5 acts as a tumor suppressor in colorectal cancer

  • These contradictory roles may reflect tissue-specific functions and interaction networks

• Consider Molecular Pathways:

  • In CRC, RBM5 binds to PTEN mRNA, stabilizes its expression, and inhibits the PI3K/AKT pathway

  • In AML, RBM5 affects genes involved in HSC self-renewal, metabolism, and leukemia development (GATA2, MSI2, MYCN, CD44, GAS2, HOXB4, and HOXB6)

  • Pathway differences may explain the opposing functions

• Evaluate Experimental Approaches:

  • Overexpression vs. knockdown studies may reveal different aspects of function

  • In vitro vs. in vivo models may show different outcomes

  • Compare results from similar experimental designs across studies

• Genetic Background Considerations:

  • RBM5's function may depend on the mutational landscape of the cancer being studied

  • In AML, RBM5 expression was significantly higher in specific genetic subtypes (CEBPA mutated, NPM1-mutated and KMT2A-r)

• Integrated Analysis:

  • Combine expression data, functional studies, and clinical outcomes

  • When possible, analyze both RBM5 and RBM10 expression, as they may cross-regulate each other

  • Consider RBM5 expression in the context of disease progression and patient outcomes

What controls should I use in RBM5 antibody validation studies?

For rigorous RBM5 antibody validation:

• Negative Controls:

  • CRISPR/Cas9 knockout samples: Successfully used in MOLM13, THP1, and OCIAML2 cells

  • RNA interference: Two validated shRNAs (RBM5: sh#1 and sh#2) efficiently suppress RBM5 expression

  • Peptide competition: Pre-incubate antibody with the immunizing peptide

  • Isotype control: Use same species IgG at equivalent concentration

• Positive Controls:

  • Overexpression: pcDNA3.1-RBM5 construct validated in SW480 and HCT116 cells

  • Tissue controls: Lung tissue (high RBM5 expression) , leukemia samples (high expression)

  • Cell lines: HeLa cells show good expression for Western blot

• Specificity Controls:

  • Cross-reactivity assessment: Test on closely related proteins (RBM10)

  • Multiple antibodies: Use antibodies targeting different epitopes of RBM5

  • Rescue experiments: Use sgRNA-resistant RBM5 cDNA to restore expression

• Method-Specific Controls:

  • WB: Molecular weight markers, loading controls (β-actin, GAPDH)

  • IHC/IF: Tissue with known expression pattern, autofluorescence controls

  • IP: Input control, IgG control, non-target protein control

  • RIP: Input RNA, IgG control, RNA quality assessment