srrm4 Antibody

What is SRRM4 and why is it an important research target?

SRRM4 (Serine/arginine repetitive matrix 4) is a nuclear protein of 611 amino acids (68.6 kDa) predominantly expressed in neuronal tissues. It functions as a splicing activator and is a member of the NSR100 protein family involved in cell differentiation and mRNA processing, particularly microexon splicing .

SRRM4 is significant for researchers because:

• It regulates microexons (3-27 nucleotides in length) that are predominantly expressed in neuronal tissues

• It plays a critical role in neuronal development and differentiation through alternative splicing mechanisms

• It has been implicated in Small Cell Lung Cancer (SCLC) pathogenesis, where it is abnormally expressed at high levels

• It may act as a proliferation brake in tumors, giving cancer cells a selective advantage when its function is suppressed

What are the optimal applications for SRRM4 antibodies in research?

SRRM4 antibodies have been validated for multiple experimental techniques, with varying degrees of effectiveness depending on the specific research question:

For researchers studying neural tissues, IHC applications consistently yield reliable results when detecting SRRM4 in paraffin-embedded sections at dilutions of 1:50-1:200 . Western blotting provides robust detection of the 68.6 kDa protein and can be particularly useful for confirming specificity when evaluating SRRM4 knockdown experiments .

How should different SRRM4 antibody formats be selected for specific research applications?

The selection of an appropriate SRRM4 antibody format should be guided by the experimental design and research objectives:

For researchers investigating SRRM4 in complex neuronal tissues where signal amplification is crucial, biotin-conjugated antibodies provide superior sensitivity. In contrast, HRP-conjugated formats are advantageous for quantitative assays where direct enzyme activity measurement is desired .

What are the key methodological considerations when using SRRM4 antibodies for cancer research?

When investigating SRRM4's role in cancer, particularly SCLC, researchers should consider:

• Tissue culture conditions affect SRRM4 expression: Studies show that culturing SCLC cells on Matrigel induces SRRM4 expression and corresponding downstream targets. This should be controlled for when designing experiments .

• Heterogeneity of SRRM4 expression: The degree of SRRM4 expression varies within SCLC cells, potentially contributing to tumor heterogeneity. Using antibodies with high sensitivity is critical .

• Combined approach with miRNA biomarkers: SRRM4 antibody detection can be complemented with miRNA analysis (particularly miR-4516) for a more comprehensive assessment of SRRM4 activity and therapeutic response .

• Validation of knockdown experiments: When testing SRRM4-targeting therapeutics (such as antisense oligonucleotides), antibody-based validation is essential to confirm protein reduction. Dose-dependent analysis shows correlation between SRRM4 reduction and cell viability .

How do SRRM4 antibodies perform in neuronal development research?

For researchers studying neuronal development:

• Expression pattern detection: SRRM4 antibodies enable visualization of the protein's dynamically changing expression pattern during development. In zebrafish studies, SRRM4 was detected in diverse cell types and developmental stages .

• Mutant validation: Antibodies are crucial for confirming the efficacy of CRISPR-based mutagenesis of SRRM4. This is particularly important given the discrepancies observed between morpholino knockdown and genetic mutation phenotypes .

• Subcellular localization: SRRM4 antibodies help confirm the protein's nuclear localization, which is critical for its function in alternative splicing regulation .

• Correlation with neuronal markers: SRRM4 antibody staining can be paired with neuronal markers to study the relationship between SRRM4 expression and neuronal differentiation or circuit formation .

What protocols optimize SRRM4 antibody performance in RNA immunoprecipitation experiments?

For researchers investigating SRRM4's interaction with target RNAs:

• Nuclear extract preparation: Since SRRM4 is a nuclear protein, optimal RNA immunoprecipitation requires careful nuclear extraction protocols. Studies have successfully used FLAG epitope-tagged SRRM4 in Neuro2A cells .

• Immunoprecipitation conditions:

  • Use antibodies to FLAG (M2) for tagged SRRM4 constructs

  • Perform immunoblot analysis to confirm precipitation

  • Use RT-PCR analysis with specific primer sets to detect RNA fragments

  • Include controls with mutated binding motifs (e.g., TGC motif mutations)

• Quantification approach: Quantify pre-mRNA bound to SRRM4 to evaluate binding efficiency. This approach has been validated for studying SRRM4's interaction with protrudin pre-mRNA and the essential UGC motif .

How can researchers troubleshoot discrepancies between SRRM4 antibody-based detection methods?

When encountering inconsistencies between different SRRM4 antibody-based detection methods:

• Epitope accessibility issues:

  • Different antibodies target different regions of SRRM4 (e.g., aa 270-319, aa 400-479)

  • Post-translational modifications like phosphorylation can affect epitope recognition

  • Fixation methods can impact epitope accessibility, particularly in IHC applications

• Validation strategy:

  • Use multiple antibodies targeting different epitopes

  • Include positive and negative controls (tissues with known expression patterns)

  • Compare with mRNA expression data when possible

  • Validate with knockout/knockdown samples

• Cross-reactivity assessment:

  • Test antibodies against known SRRM4 orthologs when working with different species

  • Consider the high conservation of SRRM4 across vertebrates when interpreting results

  • Be aware of potential cross-reactivity with other SRRM family members

What are the latest methodological advances in targeting SRRM4 for therapeutic applications?

Recent research has developed approaches where SRRM4 antibodies play a crucial role in validation:

• Gapmer antisense oligonucleotide (gASO) development:

  • gASOs targeting SRRM4 mRNA have shown efficacy in SCLC treatment

  • SRRM4 antibodies are essential for confirming protein reduction

  • The most effective gASOs contain artificial nucleic acids (LNA, AmNA) at both ends

• miRNA biomarker discovery:

  • miR-4516 has been identified as a potential biomarker for SRRM4 activity

  • Found in exosomes in the blood of SCLC patients

  • Correlates with gASO treatment efficacy

• Therapeutic efficacy assessment:

  • SRRM4 antibody-based Western blot shows dose-dependent protein reduction

  • Cell viability correlates with SRRM4 reduction

  • Tumor reduction in mouse models can be monitored alongside SRRM4 levels

How can researchers evaluate the specificity of SRRM4 antibodies across different species?

SRRM4 is highly conserved across vertebrates, with reported orthologs in mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken . Researchers should:

• Compare sequence homology:

  • Identify the exact epitope recognized by the antibody

  • Align with ortholog sequences to predict cross-reactivity

  • Pay special attention to the conserved eMIC domain, which is critical for function

• Experimental validation approach:

  • Test with recombinant proteins from different species

  • Use tissues from different species with known expression patterns

  • Include appropriate controls (knockout/knockdown samples)

• Species-specific considerations:

  • Human SRRM4 antibodies have shown reactivity with mouse and rat samples

  • For zebrafish studies, using human antibodies requires careful validation due to evolutionary distance

  • Expression patterns may differ between species (e.g., dynamic expression in zebrafish neural development)

What are the key differences between basic and advanced SRRM4 antibody applications in research?

Advanced applications require more sophisticated experimental design and often combine multiple techniques. For example, studying SRRM4's role in microexon inclusion involves:

• Antibody-based detection of SRRM4 protein levels

• Computational analysis of exon inclusion levels (PSI - percent spliced in)

• Statistical comparison between tumor and normal samples

• Integration with databases like VastDB for comprehensive splicing analysis

This integrated approach has revealed that SRRM4 silencing suppresses microexon inclusion and promotes tumor growth, suggesting its role as a tumor suppressor .

How can researchers accurately quantify SRRM4 expression using antibody-based methods?

For precise quantification of SRRM4 expression:

• Western blot quantification:

  • Use housekeeping proteins (e.g., β-actin) as loading controls

  • Apply densitometric analysis with appropriate software

  • Include standard curves with recombinant SRRM4 protein for absolute quantification

• ELISA optimization:

  • Develop sandwich ELISA using two antibodies targeting different epitopes

  • Calibrate with recombinant SRRM4 protein standards

  • Consider competitive ELISA for samples with complex matrices

• Image analysis for IHC/IF:

  • Use digital image analysis software for quantitative assessment

  • Establish consistent staining protocols with appropriate controls

  • Account for background staining and autofluorescence

• RT-qPCR correlation:

  • Correlate protein levels with mRNA expression for validation

  • Particularly important when studying splicing regulation

  • Consider post-transcriptional regulation effects

What methodological approaches help resolve contradictions between SRRM4 knockout phenotypes and antibody-based studies?

Researchers have observed discrepancies between morpholino knockdown and CRISPR-generated mutant phenotypes in SRRM4 studies. To resolve these contradictions:

• Multiple antibody validation:

  • Use antibodies targeting different epitopes to confirm protein absence/presence

  • Verify specificity with appropriate controls

• Verification of complete knockout:

  • Confirm mutation at DNA level (sequencing)

  • Verify protein absence with Western blot

  • Check for truncated protein products that may retain partial function

• Genetic compensation assessment:

  • Analyze RNA-seq data for changes in gene expression of related splicing factors

  • Look specifically for upregulation of genes that could compensate for SRRM4 function

  • Consider the possibility of nonsense-mediated decay triggering compensation

• G0 crispant vs. stable line comparison:

  • Compare phenotypes between G0 crispants (less likely to show compensation) and stable lines

  • Use brain morphometric analyses to detect subtle phenotypic changes

Research has shown that while G0 SRRM4 crispants exhibit subtle brain morphology changes (particularly in the optic tectal neuropil), stable mutant lines showed minimal splicing alterations, suggesting possible compensation mechanisms .

How can researchers optimize SRRM4 antibody selection for studying microexon splicing regulation?

For researchers investigating SRRM4's role in microexon splicing:

• Epitope consideration:

  • Select antibodies recognizing functional domains, particularly the eMIC domain

  • Ensure the epitope is not affected by splicing-related conformational changes

• Combined methodological approach:

  • Use antibodies to confirm SRRM4 expression/depletion

  • Implement computational pipelines (e.g., vast-tools) to quantify exon inclusion levels

  • Perform statistical comparisons between experimental conditions

• Data integration strategy:

  • Correlate SRRM4 protein levels with PSI (percent spliced in) values

  • Analyze across multiple tissue types to identify tissue-specific effects

  • Upload results to public databases (e.g., VastDB) for broader analysis

This approach has been successfully used to demonstrate that SRRM4 silencing suppresses microexon inclusion in tumors, with potential implications for cancer progression .

What are the critical experimental controls when using SRRM4 antibodies in research?

To ensure reliable results with SRRM4 antibodies, researchers should include:

• Positive controls:

  • Tissues with known high SRRM4 expression (neuronal tissues)

  • Cell lines with confirmed SRRM4 expression (e.g., Neuro2A, SCLC lines)

  • Recombinant SRRM4 protein or overexpression systems

• Negative controls:

  • SRRM4 knockout/knockdown samples

  • Non-neuronal tissues with minimal SRRM4 expression

  • Secondary antibody-only controls for background assessment

• Specificity controls:

  • Pre-absorption with immunogenic peptide

  • Comparison with alternative antibodies targeting different epitopes

  • Competition assays with recombinant protein

• Technical controls:

  • Loading controls for Western blots (β-actin)

  • Housekeeping gene staining for IHC

  • Environmental condition standardization (temperature, pH)

Proper controls are particularly important when studying SRRM4 in cancer research, where expression levels may vary widely between different cell populations within the same tumor .

How can researchers design experiments to investigate the relationship between SRRM4 and alternative splicing using antibodies?

To study SRRM4's role in alternative splicing:

• RNA immunoprecipitation (RIP) approach:

  • Use FLAG-tagged SRRM4 for immunoprecipitation

  • Perform RT-PCR analysis with primers flanking alternatively spliced exons

  • Quantify pre-mRNA bound to SRRM4

  • Include controls with mutated binding motifs (e.g., UGC/TGC motifs)

• SRRM4 modulation experiments:

  • Knockdown SRRM4 using siRNA or gASOs

  • Overexpress wild-type or mutant SRRM4

  • Verify protein levels with Western blot

  • Analyze splicing changes using RT-PCR or RNA-seq

• Target validation:

  • Confirm direct SRRM4 binding to target pre-mRNAs

  • Investigate specific motifs recognized by SRRM4 (e.g., UGC motif)

  • Analyze microexon inclusion rates using computational tools

  • Correlate protein levels with splicing outcomes