rbm42 Antibody

What is RBM42 and why is it important to study?

RBM42 (RNA Binding Motif Protein 42) is a multi-functional RNA-binding protein that plays critical roles in post-transcriptional regulation of gene expression. Recent studies have revealed its significance in:

• Modulating both splicing and translation of target genes, particularly during DNA damage response

• Regulating p53-responsive genes, especially CDKN1A/p21

• Contributing to pre-mRNA splicing through association with the spliceosome complex

• Participating in cellular stress responses, cell cycle regulation, and apoptosis

The protein contains one RNA recognition motif (RRM) at its C-terminal region, which is crucial for its binding to RNA targets. RBM42 is increasingly recognized as important in development and disease, with mutations linked to neurodevelopmental disorders and altered expression observed in various cancers .

What applications are RBM42 antibodies suitable for?

Based on validated research applications, RBM42 antibodies have been successfully used in:

• Western blotting (WB): Primary application with clear detection of RBM42 at 65-70 kDa

• Immunoprecipitation (IP): Effective for protein-protein interaction studies

• Immunohistochemistry (IHC): For tissue localization studies

• Enzyme-linked immunosorbent assay (ELISA): For quantitative detection

• Chromatin immunoprecipitation (ChIP): When using tagged versions (e.g., FLAG-tagged RBM42)

Most commercially available antibodies have been primarily validated for Western blotting, with different levels of validation for other applications depending on the specific antibody.

What is the expected molecular weight of RBM42 in Western blots?

While the calculated molecular weight of RBM42 is approximately 50 kDa based on its amino acid sequence, it consistently appears at 65-70 kDa in Western blots across multiple studies and antibodies . This discrepancy between calculated and observed molecular weight may be attributed to:

• Post-translational modifications

• The presence of multiple splicing isoforms (two bands are often observed in Western blots)

• Protein structure affecting migration in SDS-PAGE

Researchers should expect to see one or two bands in the 65-70 kDa range when performing Western blotting, regardless of the cell or tissue type being analyzed.

How should RBM42 antibodies be optimized for Western blotting?

For optimal Western blotting results with RBM42 antibodies:

• Recommended dilution ranges: 1:500-1:3000, with most protocols suggesting starting at 1:1000

• Sample preparation: NETN lysis buffer has been successfully used for preparing cell lysates

• Protein loading: 25-50 μg of total protein per lane is typically sufficient for detection

• Expected bands: Prepare to visualize one or two bands at 65-70 kDa

• Controls: Include positive control lysates from cells known to express RBM42 (e.g., HeLa, HEK-293T, NIH/3T3)

It's advisable to titrate the antibody concentration for your specific experimental conditions, as the optimal dilution may vary depending on the expression level of RBM42 in your samples and the detection method used.

How can I validate RBM42 antibody specificity for my research?

To ensure antibody specificity for RBM42 in your experimental system:

• Knockout validation: Compare RBM42 antibody staining between wild-type and RBM42 knockout/knockdown samples (using siRNA or CRISPR)

• Overexpression validation: Test detection of overexpressed tagged RBM42 (e.g., FLAG-RBM42-WT) alongside endogenous protein

• Multiple antibody comparison: Use antibodies targeting different epitopes of RBM42 to confirm consistent detection patterns

• Peptide competition: Pre-incubate antibody with the immunizing peptide to confirm signal reduction

• Cross-species reactivity: Test expected cross-reactivity in samples from different species based on epitope conservation

The search results describe experimental approaches where RBM42 knockdown by siRNA was used to validate antibody specificity, showing clear reduction in signal intensity proportional to the knockdown efficiency .

What are the considerations for immunoprecipitation with RBM42 antibodies?

For successful immunoprecipitation of RBM42:

• Lysis conditions: NETN buffer (150 mM NaCl, 20 mM Tris, 1% Triton X-100) supplemented with protease inhibitor cocktail has been successfully used

• Antibody amount: Approximately 1-5 μg of antibody per mg of protein lysate

• Cross-reactivity concerns: Validate the specificity of co-immunoprecipitated proteins by reciprocal IP or other methods

• Tagged versions: FLAG-tagged RBM42 can be used for more specific immunoprecipitation with anti-FLAG antibodies

• RNA-protein interactions: For studying RBM42-RNA interactions, CLIP (Crosslinking and Immunoprecipitation) has been successfully employed

For studying RBM42 interactome, researchers have successfully used proximity labeling approaches with APEX2-tagged RBM42 followed by mass spectrometry analysis .

How can I study RBM42's role in RNA splicing and translation using antibodies?

To investigate RBM42's dual role in splicing and translation regulation:

For splicing analysis:

• Perform RBM42 knockdown/knockout experiments using siRNA or CRISPR-Cas9

• Analyze alternative splicing events through RT-PCR or RNA-seq

• Validate direct interactions with splicing machinery components through co-immunoprecipitation with RBM42 antibodies

• Employ CLIP-seq to identify RBM42 binding sites in pre-mRNAs

For translation regulation studies:

• Combine RBM42 knockdown with polysome profiling to assess translational efficiency of target mRNAs

• Use RBM42 antibodies for immunoprecipitation followed by RNA isolation to identify bound mRNAs

• Perform ribosome fractionation experiments to analyze the association of RBM42 with translating ribosomes

• Assess nascent protein synthesis using methods like O-propargyl puromycin (OPP) assay

Recent research has demonstrated that RBM42 binds to the CDKN1A/p21 mRNA and affects both its splicing and translation during DNA damage response. Similar approaches can be applied to study other potential RBM42 targets .

How can RBM42 antibodies be used to study its role in DNA damage response?

To investigate RBM42's function in the DNA damage response pathway:

• Cellular localization changes: Perform immunofluorescence with RBM42 antibodies before and after DNA damage induction (e.g., etoposide/VP16 treatment, ionizing radiation)

• Protein-protein interactions: Use co-immunoprecipitation with RBM42 antibodies to identify interaction partners that specifically form during DNA damage

• Chromatin association: Perform chromatin fractionation followed by Western blotting with RBM42 antibodies

• Dynamic complex formation: Combine with proximity ligation assay (PLA) to visualize interactions between RBM42 and DNA damage response factors

• Functional correlation: Assess γH2AX levels (DNA damage marker) in RBM42-depleted cells compared to controls

Research has shown that RBM42 depletion leads to increased γH2AX levels, suggesting its involvement in genome stability maintenance. Its cytoplasmic localization also increases following DNA damage, indicating a potential shift in its functional roles during stress response .

What experimental approaches can resolve contradictory data about RBM42 function?

When facing conflicting results regarding RBM42 function:

• Cell type-specific effects: Compare RBM42 function across different cell types using the same antibody and experimental conditions

• Isoform-specific analysis: Design experiments to distinguish between different RBM42 splicing variants, potentially using isoform-specific antibodies

• Dose-dependent effects: Evaluate RBM42 function at different expression levels using partial knockdown and overexpression approaches

• Context-dependent activity: Compare RBM42 function under different cellular conditions (normal growth, DNA damage, other stresses)

• Domain-specific functions: Use domain deletion mutants (e.g., RBM42 lacking the RRM domain) to dissect the contributions of different protein regions

The literature suggests that RBM42 has context-dependent functions, acting as both a splicing regulator and translation modulator. These dual roles may explain apparently contradictory observations in different experimental settings .

How do I address non-specific bands when using RBM42 antibodies?

When encountering non-specific bands in Western blots with RBM42 antibodies:

• Optimize blocking conditions: Test different blocking agents (5% BSA vs. non-fat milk) and incubation times

• Adjust antibody dilution: Further dilute primary antibody if background is high

• Increase washing stringency: Add 0.1-0.3% Tween-20 to wash buffers and increase washing duration

• Use alternative antibodies: Compare results with antibodies targeting different epitopes of RBM42

• Confirm with controls: Include RBM42 knockdown/knockout samples as negative controls

• Consider cross-reactivity: Check if non-specific bands persist in RBM42-depleted samples, which would indicate cross-reactivity with other proteins

The observed molecular weight discrepancy (65-70 kDa vs. calculated 50 kDa) is consistent across multiple antibodies and therefore likely represents the true migration pattern of RBM42 rather than non-specific binding .

What are the best approaches for studying RBM42-RNA interactions?

For investigating interactions between RBM42 and its RNA targets:

• CLIP techniques:

  • Standard CLIP-qPCR has been successfully used to demonstrate RBM42 binding to CDKN1A mRNA

  • Enhanced CLIP (eCLIP) can identify genome-wide binding sites with higher resolution

  • Individual-nucleotide resolution CLIP (iCLIP) may reveal precise binding motifs

• In vitro binding assays:

  • Purified GST-RBM42 fusion protein can be used for RNA binding assays

  • Electrophoretic mobility shift assays (EMSA) with labeled RNA fragments

• Functional validation:

  • Mutagenesis of predicted binding sites followed by binding and functional assays

  • RNA structure analysis (e.g., DMS-Seq) to identify RBM42-dependent structural changes in target RNAs

Research has demonstrated that the RRM domain of RBM42 is essential for its RNA binding activity, as deletion mutants lacking this domain fail to bind target RNAs like CDKN1A .

How can I distinguish between RBM42's direct and indirect effects in functional studies?

To differentiate between direct and indirect effects of RBM42:

• Complementation experiments:

  • Rescue experiments using wild-type vs. binding-deficient RBM42 mutants

  • Domain-specific mutants to separate splicing vs. translation functions

• Temporal analysis:

  • Acute vs. chronic depletion using inducible knockdown/knockout systems

  • Time-course experiments following RBM42 manipulation

• Direct binding correlation:

  • Compare RNA binding (by CLIP) with functional effects on splicing and translation

  • Quantitative analysis of binding strength vs. functional impact

• Mechanistic dissection:

  • Structure-function analysis using domain mutants

  • Identification of co-factors required for specific functions

Studies have identified that RBM42 interacts with factors like RBM4 that antagonize its effects on splicing, providing mechanistic insights into its direct functions versus broader pathway effects .

How can RBM42 antibodies be used to study its role in cancer biology?

For investigating RBM42's potential contributions to cancer development and progression:

• Expression analysis:

  • Compare RBM42 protein levels between tumor and normal tissues using immunohistochemistry

  • Correlate expression with clinical parameters and patient outcomes

• Functional studies in cancer models:

  • Manipulate RBM42 expression in cancer cell lines and assess effects on proliferation, migration, and survival

  • Analyze changes in splicing and translation of cancer-relevant genes

• Therapeutic targeting assessment:

  • Use RBM42 antibodies to monitor protein expression following treatment with potential therapeutics

  • Identify potential biomarkers of response to therapy

• Mechanistic investigations:

  • Study RBM42's role in translating specific oncogenes like MYC in cancer cells

  • Investigate its contribution to stress response pathways in tumor microenvironments

Recent research indicates that RBM42 is overexpressed in various human cancers, including pancreatic ductal adenocarcinoma (PDAC), where it selectively regulates the translation of MYC and other pro-oncogenic transcripts, making it a potential therapeutic target .

What approaches are recommended for studying RBM42 in neurodevelopmental disorders?

Given the association between RBM42 mutations and neurodevelopmental disorders:

• Patient-derived models:

  • Generate induced pluripotent stem cells (iPSCs) from patients with RBM42 mutations

  • Differentiate into neurons and assess RBM42 expression and localization using antibodies

• Animal models:

  • Analyze RBM42 expression patterns in the developing brain using immunohistochemistry

  • Study phenotypes of RBM42 mutant mouse models (Q94* and A436T variants have been generated)

• Molecular consequences:

  • Assess splicing and translation of neurodevelopmentally important genes

  • Compare RNA binding profiles between wild-type and mutant RBM42

• Rescue experiments:

  • Test whether wild-type RBM42 expression can rescue phenotypes in mutant models

  • Design therapeutic approaches targeting affected downstream pathways

Research has identified biallelic variants in RBM42 causing a multisystem disorder with neurodevelopmental features, and mouse models with corresponding mutations have been generated to study this condition .

How should I design experiments to study post-translational modifications of RBM42?

To investigate potential post-translational modifications (PTMs) of RBM42:

• PTM-specific detection:

  • Use phospho-specific antibodies if available, or general phospho-detection after immunoprecipitation

  • Perform immunoprecipitation followed by mass spectrometry analysis

• Functional impact assessment:

  • Compare wild-type RBM42 with mutation of potential modification sites

  • Analyze changes in RBM42 localization, binding partners, or RNA targets following stress or stimuli

• Regulatory enzyme identification:

  • Screen for kinases, phosphatases, or other enzymes that may modify RBM42

  • Perform co-immunoprecipitation to confirm interactions

• Modification dynamics:

  • Monitor changes in RBM42 modifications during cell cycle progression or stress response

  • Correlate modifications with functional changes in splicing or translation activity

The discrepancy between calculated (50 kDa) and observed (65-70 kDa) molecular weights of RBM42 suggests potential post-translational modifications that could be functionally important but remain largely unstudied .