RBM10 Antibody

What is RBM10 and why is it relevant to study?

RBM10 (RNA-binding motif protein 10) is a 930 amino acid protein involved in post-transcriptional processing, primarily mRNA splicing. It is localized in the extranucleolar nucleoplasm within nuclear domains that dynamically change structure . RBM10 has gained significant research interest due to its involvement in tumor suppression, apoptosis regulation, and alternative splicing. Mutations in RBM10 are associated with TARP syndrome (an X-linked lethal disorder with developmental defects) and are frequently found in lung adenocarcinoma (LUAD) and renal cell carcinoma .

What molecular weight should I expect when detecting RBM10 by Western blot?

RBM10 has a calculated molecular weight of 104 kDa, but is typically observed at 110-135 kDa in Western blots. Specifically, commercial antibodies detect bands at 110 and 135 kDa (Cell Signaling Technology) or 100-120 kDa (Proteintech) . This variation reflects the existence of multiple isoforms (variants 1-5) resulting from alternative splicing events in exons 4 and 10 . When troubleshooting Western blots, researchers should verify which isoforms their antibody targets and optimize separation conditions for these higher molecular weight proteins.

How do I select the appropriate RBM10 antibody for my specific application?

Selection should be based on:

• Application compatibility: Verify antibody validation for your specific application (WB, IP, IHC, IF)

• Species reactivity: Common RBM10 antibodies react with human, mouse, and rat, but cross-reactivity varies

• Epitope location: N-terminal vs full-length antibodies detect different isoforms

• Clonality: Monoclonal antibodies (like E6I1R from Cell Signaling) offer better lot-to-lot consistency than polyclonal options

For example, Proteintech's 14423-1-AP has been validated for multiple applications (WB, IHC, IF-P, IP, CoIP, RIP) with human, mouse, and rat samples , while Cell Signaling's rabbit monoclonal E6I1R (#30774) is recombinant-derived for superior lot-to-lot consistency .

What are the optimal conditions for Western blot detection of RBM10?

For optimal Western blot detection of RBM10:

How can I optimize immunoprecipitation experiments using RBM10 antibodies?

For successful RBM10 immunoprecipitation:

• Use 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate (Proteintech recommendation)

• Cell Signaling antibodies require 1:50 or 1:100 dilution for immunoprecipitation

• Mouse brain tissue has been validated as a reliable sample for IP experiments

• For co-immunoprecipitation (CoIP) experiments investigating RBM10's protein interaction partners, include RNase treatment controls to distinguish RNA-dependent from direct protein-protein interactions

• For RIP (RNA immunoprecipitation) experiments, optimize crosslinking conditions to capture transient RNA-protein interactions

What controls should be included when performing immunostaining with RBM10 antibodies?

Essential controls for RBM10 immunostaining include:

• Positive tissue control: Use human cerebellum, prostate, or small intestine tissues, which show moderate to strong nuclear positivity in glandular cells

• Negative control: Primary antibody omission or isotype control

• RBM10 knockdown/knockout validation: Critical for confirming specificity, especially since multiple published studies have utilized RBM10 KD/KO controls

• Subcellular localization verification: RBM10 should show nuclear localization with extranucleolar staining pattern

• Competing peptide blocking: To confirm epitope specificity

How can RBM10 antibodies be used to investigate its role in cancer progression and immunotherapy response?

RBM10 antibodies enable several investigative approaches in cancer research:

• Tumor tissue profiling: IHC analysis of RBM10 expression in tumor vs. normal tissue can identify correlation with clinical features. RBM10 deficiency in LUAD is associated with higher tumor mutation burden (TMB) and improved immunotherapy response markers .

• Alternative splicing analysis: RBM10 primarily promotes exon exclusion from target pre-mRNAs. Researchers can combine RBM10 IP with RNA-seq to identify aberrantly spliced targets in cancer.

• Immune cell infiltration correlation: RBM10 deficiency correlates with increased infiltration of myeloid dendritic cells, macrophages, neutrophils, and CD8+ T cells in LUAD . IHC multiplex staining can reveal relationships between RBM10 expression and tumor immune microenvironment.

• Immunotherapy biomarker assessment: RBM10 deficiency correlates with higher expression of immune checkpoint molecules (PD-L1, TIM-3) and favorable immunotherapy response predictors (higher IFNG expression, MSI score) .

How do RBM10 variants differ functionally, and how can antibodies distinguish between them?

RBM10 has multiple splice variants resulting from:

• Inclusion/skipping of exon 4 (encoding 77 amino acids)

• Alternative 5' splice site selection in exon 10 (±Val codon)

• Internal 5' splice site selection in exon 1

These generate variants v1-v5, resulting in protein isoforms 1-5. To distinguish:

• Use isoform-specific antibodies targeting unique regions (e.g., exon 4-specific antibodies)

• Combine with molecular weight analysis (Western blot shows distinct bands at 100-135 kDa)

• For advanced applications, use IP followed by mass spectrometry to identify specific variants

• When studying variant-specific functions, verify which isoforms your antibody detects, as functional differences between variants have been reported

How can I design experiments to resolve contradictory findings about RBM10's role as tumor suppressor versus oncogene?

The literature contains some contradictory findings about RBM10's role in cancer:

To resolve these contradictions:

• Use multiple validated antibodies to confirm expression levels

• Perform isoform-specific analysis (different variants may have opposing functions)

• Analyze tissue/cell type specificity (RBM10 function may be context-dependent)

• Combine RBM10 knockdown with rescue experiments using specific variants

• Assess post-translational modifications that may alter function

• Consider heterogeneity within tumor samples (microdissection may reveal distinct expression patterns)

Why might I observe multiple bands or inconsistent molecular weights when detecting RBM10?

Multiple bands or unexpected molecular weights may occur due to:

• Isoform diversity: RBM10 has multiple splice variants (v1-v5) with predicted molecular weights ranging from 100-135 kDa

• Post-translational modifications: RBM10 undergoes phosphorylation and ubiquitination

• Proteolytic degradation: Sample preparation issues may cause degradation

• Non-specific binding: Some antibodies may cross-react with related RNA-binding proteins

Troubleshooting approaches:

• Use freshly prepared samples with protease inhibitors

• Optimize sample buffer composition and denaturing conditions

• Compare results with multiple antibodies recognizing different epitopes

• Include knockout/knockdown controls to confirm specificity

What are the key considerations for storing and handling RBM10 antibodies for maximum performance?

For optimal RBM10 antibody performance:

How can I validate RBM10 antibody specificity for confident interpretation of experimental results?

Comprehensive validation approaches include:

• Genetic controls: Test antibody in RBM10 knockout/knockdown models

• Multiple antibody comparison: Use antibodies targeting different epitopes

• Tissue/cell validation: Test in tissues with known RBM10 expression patterns

• Peptide competition: Pre-incubate antibody with immunizing peptide

• Molecular weight verification: Confirm expected size by Western blot

• Subcellular localization: Verify nuclear localization in immunofluorescence

• Species cross-reactivity: Confirm reactivity with intended species

• Application-specific validation: Validate separately for each application (WB, IP, IHC, IF)

For RBM10 specifically, human brain tissue is recommended for WB validation, while mouse brain tissue works well for IP validation . Cerebellum, prostate, and small intestine tissues show reliable immunostaining patterns .

How can RBM10 antibodies be utilized in studies of its role in immunotherapy response prediction?

Recent research has revealed RBM10 deficiency in lung adenocarcinoma correlates with enhanced anti-tumor immunity . Researchers can:

• Use RBM10 antibodies to stratify patient samples for correlation with immunotherapy response

• Combine with multiplex IHC to analyze relationships between RBM10 expression and immune cell infiltration

• Develop predictive biomarker panels including RBM10 status alongside TMB, HLA expression, and immune checkpoint levels

• Investigate mechanistic connections between RBM10-regulated splicing events and immune evasion pathways

• Perform RBM10 IP followed by RNA-seq to identify alternatively spliced immune-related transcripts

What approaches can be used to study RBM10's role in splicing regulation at the cellular level?

To investigate RBM10's splicing activity:

• Combine RBM10 IP with sequencing (RIP-seq) to identify bound RNA targets

• Use CLIP techniques (cross-linking immunoprecipitation) with RBM10 antibodies to map binding sites with nucleotide resolution

• Perform RNA-seq following RBM10 depletion to identify splicing events regulated by RBM10

• Develop in vitro splicing assays with recombinant RBM10 and candidate target pre-mRNAs

• Apply live-cell imaging with fluorescently tagged RBM10 to visualize dynamic interactions with the splicing machinery