rbm24 Antibody

What is RBM24 and why is it significant in research?

RBM24 is an RNA-binding protein that regulates pre-mRNA splicing and mRNA stability, serving as a multifunctional determinant of cell fate, proliferation, apoptosis, and differentiation during development. It exhibits temporally and spatially regulated expression patterns, particularly in muscle tissue development and regeneration . RBM24 has been identified as a critical factor in myogenic differentiation, being required for myogenin expression at early stages of muscle injury and for muscle-specific pre-mRNA alternative splicing at late stages of regeneration . Recent research has also implicated RBM24 in carcinogenesis, particularly in bladder cancer progression, where higher expression correlates with poor patient survival . The multifaceted roles of RBM24 in development, regeneration, and disease make it an important target for diverse research applications, from developmental biology to cancer research.

What are the common applications for RBM24 antibodies in research?

RBM24 antibodies serve multiple experimental applications in molecular and cellular biology research. The most common validated applications include:

RBM24 antibodies are particularly valuable for studying muscle development, regeneration processes, and cancer progression models. They enable researchers to detect both endogenous RBM24 expression and track distribution patterns during cellular differentiation and tissue development .

What is the molecular weight of RBM24 and how does this impact antibody selection?

The calculated molecular weight of RBM24 is approximately 20 kDa (191 amino acids), but the observed molecular weight on Western blots ranges from 18-25 kDa . This variability could reflect post-translational modifications or tissue-specific isoform expression. When selecting an antibody, researchers should consider:

• Whether the antibody has been validated to detect the specific isoform of interest

• The target epitope location (N-terminal vs. C-terminal)

• Cross-reactivity with related RNA-binding proteins

For example, the RBM24 antibody (18178-1-AP) from Proteintech targets the full RBM24 protein and shows reactivity with human, mouse, and rat samples , while the RayBiotech antibody (102-16537) targets specifically the N-terminal region (amino acids 4-32) of human RBM24 . This epitope difference may be critical depending on your experimental model and research questions.

How should I design experiments to study RBM24 localization during muscle development?

RBM24 exhibits dynamic subcellular localization during muscle development, with translocation from cytoplasm to nucleus during myoblast differentiation . To effectively study this process:

• Time-course analysis: Establish a clear timeline for sampling during differentiation. RBM24 studies in C2C12 myoblast differentiation have revealed critical transition points that should be captured (typically days 0, 1, 3, 5, and 7 of differentiation) .

• Subcellular fractionation: Complement microscopy with biochemical fractionation to quantify nuclear vs. cytoplasmic distribution changes.

• Co-localization studies: Use dual immunofluorescence to track RBM24 in relation to known markers of:

  • Myogenic differentiation (MyoD, myogenin)

  • Nuclear structures (lamin B)

  • RNA processing bodies (P-bodies, stress granules)

• Live-cell imaging: For dynamic studies, consider using fluorescently tagged RBM24 constructs, though validation against endogenous protein behavior is essential.

A methodologically sound approach would combine IF-P (Immunofluorescence on paraffin sections) at dilutions between 1:50-1:500 with Western blot analysis of subcellular fractions to quantitatively track localization shifts during developmental progression or in response to experimental manipulations.

What controls should be included when using RBM24 antibodies for studying alternative splicing regulation?

When investigating RBM24's role in alternative splicing, rigorous controls are essential:

• Antibody validation controls:

  • Positive tissue controls (heart and skeletal muscle tissue show high endogenous expression)

  • RBM24 knockout or knockdown samples as negative controls

  • Peptide competition assays to confirm specificity

• Experimental controls for splicing studies:

  • Analysis of constitutively spliced exons unaffected by RBM24 as internal controls

  • Inclusion of related RNA-binding proteins (e.g., RBM38) to assess specificity of effects

  • Rescue experiments with wild-type RBM24 following knockdown/knockout

• Minigene splicing assays:

  • Empty vector controls when performing co-transfection experiments

  • Dose-response analysis with varying RBM24 expression levels

  • Mutational analysis of predicted RBM24 binding sites

Research by Yang et al. demonstrated that Rbm24 regulates multiple alternative splicing events essential for myogenic differentiation and muscle regeneration, including AS of Mef2d, Naca, Rock2, and Lrrfip1 . Their study employed rigorous controls, including inducible knockout models and rescue experiments, which should be considered when designing similar studies.

How can RBM24 antibodies be used to investigate tissue-specific expression patterns in muscle subtypes?

RBM24 exhibits differential expression across muscle fiber types, with enrichment in slow-twitch muscles, correlating with myogenin mRNA expression patterns . To effectively investigate tissue-specific expression:

• Comparative analysis across muscle types:

  • Use consistent antibody concentrations (1:20-1:200 for IHC ) across sample types

  • Include multiple muscle types (e.g., soleus for slow-twitch, extensor digitorum longus for fast-twitch)

  • Correlate RBM24 expression with established fiber-type markers (MHC isoforms)

• Single-fiber analysis:

  • Combine RBM24 immunostaining with fiber-type-specific markers

  • Implement quantitative image analysis to compare nuclear localization intensity

  • Consider co-staining with satellite cell markers to distinguish myonuclear from satellite cell expression

• Injury models for dynamic assessment:

  • Utilize cardiotoxin (CTX)-induced injury models to track temporal changes in RBM24 expression

  • Sample at defined timepoints (days 3, 7, 14, 28 post-injury) to capture regeneration phases

  • Compare regeneration dynamics between muscle types with different baseline RBM24 expression

Research has shown that upon injury, RBM24 is rapidly upregulated in regenerating myofibers and accumulates in the myonuclei of nascent myofibers , making antibody-based detection particularly valuable for regeneration studies.

What methodological approaches are recommended for studying RBM24's role in cancer progression?

Recent research has implicated RBM24 in cancer biology, particularly bladder cancer progression, where it forms a positive feedback loop with Runx1t1 and miR-625-5p . To investigate RBM24's role in cancer:

• Expression correlation studies:

  • Apply RBM24 antibodies for tissue microarray analysis of patient samples

  • Correlate expression with clinical outcomes and established cancer markers

  • Implement multi-parameter immunofluorescence to assess co-expression with known oncogenic pathways

• Functional studies in cancer models:

  • Use RBM24 antibodies to validate knockdown/overexpression efficiency in cancer cell lines

  • Apply RBM24 immunoprecipitation (RIP) to identify cancer-specific RNA targets

  • Combine with in vivo xenograft models to assess effects on tumor growth and metastasis

• Mechanistic investigations:

  • Examine RBM24-regulated alternative splicing events in cancer contexts

  • Investigate RBM24's impact on mRNA stability of oncogenes and tumor suppressors

  • Explore interactions with cancer-associated transcription factors and miRNAs

Research has shown that higher RBM24 and Runx1t1 levels in bladder cancer tissue correlate with poor patient survival . The RBM24/Runx1t1/TCF4/miR-625-5p positive feedback loop presents a potential therapeutic target, and antibody-based methods are essential for elucidating these complex interactions.

What are the optimal conditions for using RBM24 antibodies in different experimental systems?

Successful application of RBM24 antibodies requires optimization for specific experimental systems:

Key optimization tips:

• Always titrate antibodies for each new cell line or tissue type

• For muscle tissue, recommended antigen retrieval with TE buffer pH 9.0 has shown superior results

• For nuclear localization studies, ensure proper nuclear permeabilization

• When analyzing developmental processes, standardize fixation protocols across timepoints

How can I address cross-reactivity or specificity concerns with RBM24 antibodies?

Ensuring antibody specificity is critical for reliable RBM24 research:

• Validation strategies:

  • Use Rbm24 knockout tissues as definitive negative controls

  • Employ siRNA/shRNA knockdown to generate decreased signal controls

  • Consider using multiple antibodies targeting different epitopes of RBM24

• Cross-reactivity assessment:

  • Test antibodies on tissues with known absence of RBM24 expression

  • Check for reactivity with closely related RBM family members (especially RBM38)

  • Conduct peptide competition assays to confirm epitope specificity

• Addressing inconsistent results:

  • Different antibodies may recognize distinct isoforms or post-translationally modified variants

  • Epitope masking can occur in specific cellular contexts

  • Signal-to-noise ratio may vary across applications and tissue types

For researchers encountering specificity issues, the conditional knockout mouse models described by Yang et al. and the satellite cell-specific knockout mice provide excellent validation systems to confirm antibody specificity in muscle research contexts.

How can RBM24 antibodies facilitate investigation of alternative splicing networks in muscle regeneration?

Recent research has revealed RBM24 as a critical regulator of alternative splicing events essential for muscle regeneration . To leverage RBM24 antibodies in this research:

• Integrated multi-omics approaches:

  • Combine RBM24 RIP-seq with RNA-seq to identify direct splicing targets

  • Correlate RBM24 binding sites with splicing outcomes

  • Use RBM24 antibodies for CLIP-seq to identify precise RNA binding motifs

• Temporal dynamics analysis:

  • Track RBM24 expression and localization across regeneration timepoints

  • Correlate with emergence of muscle-specific splice variants

  • Implement pulse-chase experiments to assess protein stability during regeneration

• Single-cell applications:

  • Apply RBM24 antibodies in single-cell mass cytometry

  • Correlate RBM24 levels with cell state transitions during regeneration

  • Combine with lineage tracing to follow satellite cell differentiation

The study by Yang et al. demonstrated that Rbm24 regulates a complex network of alternative splicing events involved in multiple biological processes, including myogenesis, muscle regeneration, and muscle hypertrophy . Using RBM24 antibodies to dissect these networks temporally can provide critical insights into regeneration mechanisms.

What methodological considerations are important when investigating RBM24's dual roles in mRNA stability and alternative splicing?

RBM24 exhibits dual functionality in regulating both mRNA stability and alternative splicing , requiring thoughtful experimental design:

• Distinguishing direct from indirect effects:

  • Use RBM24 RIP followed by specific transcript quantification

  • Implement actinomycin D chase experiments to assess mRNA stability

  • Design minigene constructs to isolate splicing from stability effects

• Domain-specific functions:

  • Consider antibodies targeting specific functional domains of RBM24

  • Use truncation mutants and domain swaps to map function-specific regions

  • Correlate binding patterns with functional outcomes

• Context-dependent activity:

  • Compare RBM24 activity across differentiation states

  • Assess cooperative interactions with other RNA-binding proteins

  • Evaluate post-translational modifications that might switch functionality

Research by Yang et al. revealed that Rbm24 knockout in skeletal muscle resulted in myogenic fusion and differentiation defects with significantly delayed muscle regeneration . Mechanistically, they demonstrated RBM24's regulation of alternative splicing of Mef2d, Naca, Rock2, and Lrrfip1, which are essential for myogenic differentiation and muscle regeneration .