RBM12 Antibody

What is RBM12 and what are its known functions?

RBM12, also known as SWAN, HRIHFB2091, or KIAA0765, is a 932 amino acid nuclear protein primarily involved in RNA processing and gene expression regulation. The protein contains multiple functional domains including three RNA recognition motifs (RRMs), proline-rich regions, and transmembrane domains . RBM12 plays crucial roles in:

• Regulation of alternative splicing mechanisms

• Modulation of apoptotic pathways

• Repression of fetal hemoglobin (HbF) expression in adult erythroid cells

• Potential involvement in tumor immunity, particularly in hepatocellular carcinoma

• Regulation of β2-adrenergic receptor/cAMP signaling pathways

The protein's nuclear localization suggests it functions at the level of pre-mRNA synthesis or processing, making it an important player in transcriptional and post-transcriptional regulation . Recent research indicates that RBM12 may be implicated in schizophrenia and psychosis as a high-penetrance risk factor, expanding its significance to neuropsychiatric disorders .

What applications are RBM12 antibodies suitable for?

RBM12 antibodies, particularly the mouse monoclonal IgG2a antibody (E-6), have been validated for multiple experimental applications across various species including human, mouse, and rat samples . The primary applications include:

When designing experiments, it's important to note that RBM12 antibodies can be used for both qualitative characterization (localization, interaction partners) and quantitative analysis (expression levels in different tissues or disease states) .

How is RBM12 expression regulated across different tissues and developmental stages?

RBM12 displays broad tissue expression without specific developmental regulation between fetal and adult stages. Unlike some developmental regulators that show stage-specific expression:

• RBM12 is expressed across various tissue types, including throughout the hematopoietic system

• In the erythroid compartment, RBM12 levels are initially high and gradually decrease during terminal differentiation

• Expression levels remain relatively constant between fetal and adult erythroid stages, unlike other developmental globin regulators

• RBM12 expression in hepatocellular carcinoma is upregulated through hypomethylation mechanisms

This expression pattern suggests that RBM12's function may be primarily regulated through post-translational modifications or interaction partners rather than through developmental transcriptional control mechanisms .

How can RBM12 antibodies be utilized in cancer research, particularly for hepatocellular carcinoma studies?

RBM12 has been identified as a potential prognostic marker in hepatocellular carcinoma (HCC), with overexpression correlating with poor patient outcomes . For researchers investigating RBM12 in cancer contexts:

• Immunohistochemistry with RBM12 antibodies can be used to assess expression levels in tumor versus normal tissues, with quantification possible using standard scoring methods

• Western blotting can quantitatively measure RBM12 protein levels across different cancer cell lines or patient-derived samples

• For mechanistic studies, RBM12 antibodies can be employed in chromatin immunoprecipitation (ChIP) or RNA immunoprecipitation (RIP) assays to identify DNA/RNA targets

Experimental design should incorporate both transcriptional analysis (qRT-PCR) and protein-level analysis (western blot with RBM12 antibody) to comprehensively evaluate RBM12 dysregulation. In HCC research specifically, RBM12 antibodies have been used to demonstrate that overexpression is caused by hypomethylation and that RBM12 plays a key role in liver cancer tumor immunity .

What methodologies incorporate RBM12 antibodies for investigating hematopoietic disorders?

Recent CRISPR/Cas9 screening has identified RBM12 as a novel repressor of fetal hemoglobin (HbF), with significant implications for sickle cell disease and β-thalassemia research . When studying RBM12 in hematopoietic contexts:

• RBM12 antibodies can be used for western blot analysis to confirm CRISPR/Cas9-mediated knockout efficiency in erythroid cells

• Immunofluorescence staining with anti-RBM12 antibodies demonstrates the protein's nuclear localization in primary human erythroblasts

• Enhanced cross-linking and immunoprecipitation followed by high-throughput sequencing (eCLIP-seq) with RBM12 antibodies (e.g., sc-514259) can identify RNA binding patterns, particularly at 5' untranslated regions

For researchers investigating hemoglobinopathies, RBM12 antibodies are crucial for validating experimental models where RBM12 manipulation affects HbF levels. Studies have shown that RBM12 depletion induces HbF expression and attenuates cell sickling in erythroid cells derived from patients with sickle cell disease, suggesting therapeutic potential .

How can RBM12 antibodies be employed in RNA-binding protein research?

As RBM12 contains multiple RNA recognition motifs (RRMs), researchers investigating RNA-protein interactions can utilize RBM12 antibodies in several specialized techniques:

• eCLIP-seq protocols with anti-RBM12 antibodies (such as sc-514259) allow for high-resolution mapping of RNA-protein interaction sites, following established ENCODE protocols

• RBM12 shows preferential binding to 5' untranslated regions of transcripts, providing insight into its regulatory mechanisms

• Domain-specific studies can be conducted using truncated RBM12 constructs in conjunction with antibodies that recognize specific regions to determine which RRMs are critical for particular functions

Experimental results have demonstrated that the first of RBM12's five RRM domains is essential for its HbF repression activity, highlighting the importance of domain-specific functional analysis in RBP research .

What is the recommended protocol for using RBM12 antibodies in western blotting?

For optimal results when using RBM12 antibodies in western blotting:

• Sample preparation: Lyse cells in RIPA buffer supplemented with protease inhibitors

• Protein quantification: Use BCA or Bradford assay to ensure equal loading

• SDS-PAGE: Load 20-50 μg of protein per lane on 8-10% gels (RBM12 is approximately 102 kDa)

• Transfer: Use PVDF membrane with standard wet transfer protocols

• Blocking: 5% non-fat dry milk in TBST for 1 hour at room temperature

• Primary antibody: Dilute RBM12 antibody (e.g., sc-514259) at 1:1000 in blocking solution and incubate overnight at 4°C

• Washing: 3 × 10 minutes with TBST

• Secondary antibody: Anti-mouse IgG (1:2000, A0216) for 1 hour at room temperature

• Detection: Use chemiluminescence or fluorescence-based detection systems

• Controls: Include β-actin (1:1000, AF0003) as loading control

In hepatocellular carcinoma research, this protocol has successfully demonstrated elevated RBM12 expression in tumor tissues compared to adjacent normal tissues, correlating with poor patient prognosis .

How should RBM12 antibodies be utilized in enhanced cross-linking and immunoprecipitation followed by sequencing (eCLIP-seq)?

eCLIP-seq with RBM12 antibodies requires specific protocols to identify RNA-binding sites:

• Cross-linking: Expose cells to UV radiation (254 nm, 400 mJ/cm³) to create covalent bonds between RBM12 and bound RNA

• Cell lysis: Lyse cells and fragment RNA via controlled RNase I digestion

• Immunoprecipitation: Use anti-RBM12 antibody (sc-514259) coupled to magnetic beads (10003D)

• Washing: Perform stringent washes to remove non-specific interactions

• RNA isolation: Purify RNA from the RBM12-RNA complexes

• Library preparation: Ligate RNA barcode adapters, reverse transcribe, and prepare cDNA libraries

• Sequencing: Perform high-throughput sequencing on platforms such as NextSeq2000

• Data analysis: Align reads to reference genome and identify enriched binding motifs

This methodology has revealed that RBM12 preferentially binds to 5' untranslated regions of transcripts, providing critical insight into its mechanism of action in regulating gene expression .

What approaches can resolve contradictory results when using RBM12 antibodies?

When faced with contradictory results in RBM12 antibody experiments, consider these methodological troubleshooting approaches:

• Antibody validation: Confirm antibody specificity using positive controls (cells/tissues known to express RBM12) and negative controls (RBM12 knockout samples)

• Multiple detection methods: Cross-validate findings using different techniques (western blot, IF, qRT-PCR)

• Epitope consideration: Different antibodies may recognize different epitopes; use alternative RBM12 antibodies targeting distinct regions

• Experimental conditions: Optimize fixation, permeabilization, and blocking conditions for immunofluorescence studies

• Expression variability: Account for RBM12 expression changes during cell differentiation, particularly in erythroid cells where levels fade during terminal differentiation

• Alternative splicing: Consider the presence of multiple RBM12 isoforms that share the same coding sequence but may differ in expression patterns

When studying RBM12 in disease contexts, particularly in cancer research, it's essential to use matched normal and tumor tissues from the same patients to control for individual variation in expression levels .

How can RBM12 antibodies contribute to understanding neuropsychiatric disorders?

Recent discoveries of RBM12 as a high-penetrance risk factor for familial schizophrenia and psychosis open new research avenues:

• RBM12 antibodies can be used to examine protein expression in post-mortem brain tissue from patients with schizophrenia compared to controls

• Immunohistochemistry and immunofluorescence with RBM12 antibodies can map expression across different brain regions and cell types

• Co-immunoprecipitation with RBM12 antibodies may identify interaction partners in neuronal cells, potentially revealing disease mechanisms

• RBM12's role as a repressor of β2-AR/cAMP signaling suggests investigation of this pathway in neuropsychiatric contexts

Researchers should design experiments comparing RBM12 expression and localization in neurons derived from patient-specific induced pluripotent stem cells (iPSCs) versus control iPSCs, potentially revealing disease-specific alterations in RBM12 function or localization .

What is the potential of RBM12 as a therapeutic target for hemoglobinopathies?

The identification of RBM12 as a novel HbF repressor presents interesting therapeutic opportunities:

• RBM12 antibodies are essential tools for screening potential drug candidates that inhibit RBM12 function

• Immunoprecipitation with RBM12 antibodies followed by mass spectrometry can identify binding partners that might offer alternative therapeutic targets

• In sickle cell disease models, RBM12 depletion has been shown to increase HbF levels by >2-fold and decrease cell sickling by ~50%

• Unlike other HbF regulators, RBM12 functions independently of BCL11A, potentially offering a complementary therapeutic approach

Researchers investigating this pathway should focus on the first RRM domain and the proline-rich linker region, as these have been identified as sufficient for RBM12-mediated HbF repression. This domain specificity could guide the development of targeted therapeutics that disrupt only the HbF-regulatory function without affecting other potential RBM12 functions .