SRRM4 Antibody, HRP conjugated

What is SRRM4 and why is it significant in neuroscience research?

SRRM4 (Serine/arginine repetitive matrix protein 4) is a nuclear splicing factor specifically required for neural cell differentiation. It functions by binding directly to regulated target transcripts and promoting the inclusion of neural-specific exons in target mRNAs . With a molecular weight of 68.6 kDa and 611 amino acid residues in humans, SRRM4 is predominantly expressed in neuronal cells . Its critical role in neural-specific alternative splicing makes it an important research target for understanding neuronal development, function, and related pathologies. Recent evidence also suggests SRRM4 involvement in neuroendocrine differentiation in certain cancer types, expanding its research significance beyond neuroscience .

What advantages does HRP conjugation provide for SRRM4 antibody applications?

HRP (Horseradish Peroxidase) conjugation to SRRM4 antibodies offers several methodological advantages in research applications. The enzyme conjugation provides enhanced sensitivity through signal amplification when appropriate substrates are added, generating colorimetric, chemiluminescent, or fluorescent signals. This direct conjugation eliminates the need for secondary antibody incubation steps, reducing background signal and protocol complexity. HRP-conjugated antibodies are particularly valuable in ELISA applications, where they enable straightforward detection with standard plate readers and various substrates . The stability of HRP conjugates also allows for longer storage and consistent performance across experiments when properly maintained.

What are the key specifications to consider when selecting an HRP-conjugated SRRM4 antibody?

When selecting an HRP-conjugated SRRM4 antibody, researchers should consider several critical specifications:

• Epitope specificity: Some antibodies target specific regions of SRRM4 (e.g., AA 400-479 as described in the product datasheet) , which is important when studying different functional domains.

• Host species and clonality: Available products include rabbit polyclonal antibodies, which offer broad epitope recognition .

• Purity level: Higher purity antibodies (e.g., >95% Protein G purified) generally provide more consistent results with lower background .

• Cross-reactivity profile: Determine if the antibody cross-reacts with SRRM4 from other species if conducting comparative studies. Some antibodies show reactivity with human SRRM4 only, while others may cross-react with mouse, rat, or other species .

• Validated applications: Confirm the antibody has been validated for your specific application, such as ELISA, Western blot, or immunohistochemistry .

• Conjugate stability: Consider the shelf-life and storage requirements of the HRP conjugate to maintain enzymatic activity.

What controls are essential when using HRP-conjugated SRRM4 antibodies?

A robust experimental design with HRP-conjugated SRRM4 antibodies should include these essential controls:

• Positive tissue control: Neural tissues or neuronal cell lines known to express SRRM4, which is primarily expressed in neuronal cells .

• Negative tissue control: Samples known not to express SRRM4 or where expression has been knocked down/out through genetic manipulation.

• Isotype control: An irrelevant antibody of the same isotype (e.g., rabbit IgG) also conjugated to HRP to establish baseline non-specific binding .

• Blocking peptide control: Pre-incubation of the antibody with its specific immunogen (e.g., recombinant SRRM4 protein aa 400-479) to confirm binding specificity .

• Endogenous peroxidase control: Especially important for tissue samples, include steps to quench endogenous peroxidase activity before antibody application.

• Dilution optimization control: A dilution series of the antibody to determine optimal signal-to-noise ratio for your specific sample type.

• Substrate-only control: To assess background from the detection system alone without antibody.

What is the optimal protocol for ELISA using HRP-conjugated SRRM4 antibodies?

The optimal ELISA protocol for HRP-conjugated SRRM4 antibodies involves these methodological steps:

• Plate preparation:

  • Coat high-binding 96-well plate with capture antigen or antibody (if sandwich ELISA)

  • Incubate overnight at 4°C in appropriate coating buffer

  • Wash 3-5 times with washing buffer (PBS-T: PBS + 0.05% Tween-20)

• Blocking:

  • Block remaining binding sites with 1-5% BSA or non-fat dry milk in PBS

  • Incubate for 1-2 hours at room temperature

  • Wash 3-5 times with washing buffer

• Sample addition:

  • Add samples and standards in dilution buffer

  • Incubate for 2 hours at room temperature or overnight at 4°C

  • Wash 5 times with washing buffer

• HRP-conjugated antibody application:

  • Dilute HRP-conjugated SRRM4 antibody according to manufacturer's recommendations (typically 1:500 to 1:5000)

  • Incubate for 1-2 hours at room temperature

  • Wash 5-7 times with washing buffer

• Signal development:

  • Add appropriate HRP substrate (TMB, ABTS, or OPD)

  • Monitor color development (typically 5-30 minutes)

  • Stop reaction with stopping solution if using TMB (e.g., 2N H₂SO₄)

• Data acquisition:

  • Read absorbance at appropriate wavelength

  • Analyze data against standard curve

How should samples be prepared for optimal SRRM4 detection in neural tissues?

Optimal sample preparation for SRRM4 detection in neural tissues requires careful consideration of its nuclear localization and neural-specific expression:

• Tissue fixation options:

  • For formalin-fixed paraffin-embedded (FFPE) sections: Fix in 10% neutral-buffered formalin for 24-48 hours, followed by paraffin embedding

  • For frozen sections: Flash freeze in liquid nitrogen and section at 8-12 μm thickness

  • For cell preparations: Fix with 4% paraformaldehyde for 15-20 minutes

• Antigen retrieval for FFPE tissues:

  • Heat-mediated antigen retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Pressure cook for 10-15 minutes or microwave for 20 minutes

  • Allow gradual cooling to room temperature

• Permeabilization for nuclear antigen:

  • SRRM4 is primarily nuclear, requiring effective nuclear permeabilization

  • Use 0.1-0.3% Triton X-100 in PBS for 10-15 minutes

  • For cell preparations, 0.1% Triton X-100 for 5-10 minutes is typically sufficient

• Blocking steps:

  • Block endogenous peroxidase with 0.3-3% H₂O₂ treatment for 10-15 minutes

  • Use 5-10% normal serum (from the same species as the secondary antibody if using an indirect detection system)

  • Consider dual blocking with serum and protein blockers (e.g., 1% BSA)

• Sample handling considerations:

  • Maintain consistent section thickness across experimental groups

  • Process all comparative samples simultaneously to minimize technical variation

  • Store sections appropriately to maintain antigen integrity

What are common issues with HRP-conjugated SRRM4 antibodies and how can they be resolved?

When working with HRP-conjugated SRRM4 antibodies, researchers may encounter several common issues with specific resolution strategies:

• High background signal:

  • Cause: Insufficient blocking, too concentrated antibody, or inadequate washing

  • Solution: Optimize blocking conditions (try different blocking proteins), increase antibody dilution, and implement more stringent washing steps

• Weak or no signal:

  • Cause: Insufficient antigen, over-diluted antibody, or suboptimal antigen retrieval

  • Solution: Verify SRRM4 expression in sample (neural tissues are positive controls), decrease antibody dilution, optimize antigen retrieval conditions (temperature, buffer, duration)

• Non-specific binding:

  • Cause: Cross-reactivity with related proteins or insufficient blocking

  • Solution: Pre-absorb antibody with potential cross-reactive proteins, use more specific blocking reagents, consider monoclonal alternatives if using polyclonal antibodies

• Inconsistent results:

  • Cause: Variability in sample preparation, antibody aliquots, or detection conditions

  • Solution: Standardize all protocols, prepare fresh working dilutions for each experiment, and maintain consistent temperature and timing

• HRP activity loss:

  • Cause: Improper storage, repeated freeze-thaw cycles, or exposure to contaminants

  • Solution: Store according to manufacturer recommendations, prepare single-use aliquots, use stabilizing diluents for working solutions

How can specificity of SRRM4 antibody binding be validated in experimental systems?

Validating the specificity of SRRM4 antibody binding requires multiple complementary approaches:

• Molecular validation:

  • SRRM4 knockdown/knockout: Generate cell lines with reduced or eliminated SRRM4 expression to confirm signal reduction

  • Peptide competition: Pre-incubate antibody with immunizing peptide to block specific binding

  • Western blot: Verify detection of a single band at the expected molecular weight (68.6 kDa)

• Comparative validation:

  • Multi-antibody approach: Test multiple antibodies targeting different SRRM4 epitopes

  • Cross-species comparison: Examine detection patterns in species with known sequence conservation

  • Tissue distribution analysis: Confirm higher expression in neural tissues compared to non-neural tissues

• Functional validation:

  • Correlation with functional readouts: Compare SRRM4 detection with alternative splicing patterns of known targets

  • Co-localization studies: Verify nuclear localization consistent with its role as a splicing factor

  • Expression timing: Confirm expression patterns during neural differentiation match known SRRM4 activity patterns

• Technical validation:

  • Titration experiments: Perform serial dilutions to establish optimal concentration

  • Signal linearity assessment: Verify that signal strength correlates with known expression levels

  • Method comparison: Compare results across different detection platforms (IHC, Western blot, ELISA)

What optimization strategies can enhance detection sensitivity for SRRM4 in samples with low expression?

For enhancing detection sensitivity of SRRM4 in samples with low expression levels, consider these optimization strategies:

• Signal amplification methods:

  • Tyramide signal amplification (TSA): Implement TSA systems compatible with HRP to amplify signal 10-100 fold

  • Multi-layer detection: Use biotin-streptavidin systems with multiple HRP molecules per binding event

  • Polymer-based detection: Utilize detection systems with multiple HRP molecules conjugated to polymers

• Sample preparation enhancements:

  • Subcellular fractionation: Enrich for nuclear proteins where SRRM4 is primarily localized

  • Longer primary antibody incubation: Extend to overnight at 4°C to maximize binding

  • Optimized antigen retrieval: Systematically test different pH buffers and retrieval conditions

• Technical adjustments:

  • Reduce background: Implement extended blocking steps with specialized blocking reagents

  • Optimize substrate: Select highly sensitive substrates for HRP (e.g., enhanced chemiluminescence for Western blots)

  • Increase antibody concentration: For samples with known low expression, higher antibody concentrations may be necessary

• Detection system modifications:

  • Digital image capture: Use longer exposure times and higher sensitivity settings

  • Signal integration: Employ longer data acquisition times for spectrophotometric readings

  • Noise reduction algorithms: Apply computational methods to enhance signal-to-noise ratio

How can HRP-conjugated SRRM4 antibodies be used to study alternative splicing in neural differentiation models?

HRP-conjugated SRRM4 antibodies can be strategically employed to investigate alternative splicing in neural differentiation through several advanced approaches:

• Temporal expression profiling:

  • Track SRRM4 protein levels across neural differentiation time points

  • Correlate SRRM4 expression with neural marker appearance and alternative exon inclusion

  • Quantify changes in expression levels during critical developmental windows

• Chromatin immunoprecipitation (ChIP) applications:

  • Adapt HRP-conjugated antibodies for ChIP protocols to identify SRRM4 binding sites

  • Map binding patterns to exon-intron boundaries of alternatively spliced genes

  • Correlate binding data with RNA-seq analysis of alternative splicing events

• Co-localization studies:

  • Perform dual labeling with spliceosome components using fluorescent secondary detection

  • Visualize SRRM4 recruitment to active splicing sites during neural differentiation

  • Quantify changes in nuclear speckle morphology and composition

• Functional manipulation experiments:

  • Monitor SRRM4 protein expression following knockdown/overexpression interventions

  • Correlate protein levels with functional readouts of neural differentiation

  • Identify threshold levels required for alternative splicing regulation

• Cell type-specific analyses:

  • Compare SRRM4 expression across neural subtypes (neurons vs. glia)

  • Identify cell type-specific co-factors that interact with SRRM4

  • Correlate expression patterns with cell type-specific splicing events

What methodological approaches are recommended for studying SRRM4's role in neuroendocrine cancer progression?

Based on emerging evidence of SRRM4's role in neuroendocrine differentiation , these methodological approaches are recommended:

• Tissue microarray analysis:

  • Develop standardized immunohistochemistry protocols using HRP-conjugated SRRM4 antibodies

  • Compare expression across cancer progression stages using tissue microarrays

  • Correlate with established neuroendocrine markers (chromogranin A, synaptophysin)

• Patient-derived models:

  • Establish patient-derived xenografts or organoids representing different disease stages

  • Monitor SRRM4 expression during treatment responses and resistance development

  • Correlate expression with treatment outcomes and patient survival data

• Mechanistic studies:

  • Perform SRRM4 manipulation experiments in relevant cancer cell lines

  • Identify direct splicing targets using RNA immunoprecipitation followed by sequencing

  • Validate key targets using minigene splicing assays

• Therapeutic targeting assessment:

  • Screen for compounds that modulate SRRM4 expression or activity

  • Evaluate effects on neuroendocrine differentiation and tumor growth

  • Determine potential synergistic effects with standard-of-care treatments

• Biomarker development pipeline:

  • Optimize detection protocols for clinical sample types

  • Establish quantitative scoring systems for SRRM4 immunostaining

  • Determine sensitivity and specificity for predicting disease progression

How can HRP-conjugated SRRM4 antibodies be integrated with other technologies for comprehensive neural protein-RNA interaction studies?

Integration of HRP-conjugated SRRM4 antibodies with complementary technologies enables comprehensive protein-RNA interaction studies:

• CLIP-seq integration:

  • Use HRP-conjugated SRRM4 antibodies to validate protein expression in cells prepared for CLIP-seq

  • Correlate protein levels with binding site frequency and strength

  • Develop dual protocol workflows for simultaneous protein detection and RNA binding assessment

• Spatial transcriptomics combination:

  • Perform HRP-based SRRM4 detection on tissue sections

  • Apply spatial transcriptomics on sequential sections (e.g., Visium platform)

  • Integrate protein expression maps with spatially-resolved alternative splicing data

• Proximity ligation adaptations:

  • Combine SRRM4 antibodies with antibodies against other splicing factors

  • Visualize and quantify in situ protein-protein interactions in the splicing machinery

  • Map interaction networks across neural development or disease progression

• Live-cell applications:

  • Develop protocols for live-cell imaging of SRRM4 using antibody fragments

  • Track dynamic associations with target RNAs using MS2-tagged transcripts

  • Measure kinetics of complex assembly and disassembly during splicing events

• Mass spectrometry workflows:

  • Use HRP-conjugated antibodies for immunoprecipitation followed by mass spectrometry

  • Identify novel SRRM4 interacting partners in different neural subtypes

  • Discover post-translational modifications that regulate SRRM4 activity

How does the performance of HRP-conjugated SRRM4 antibodies compare with other detection systems?

A comprehensive comparison of HRP-conjugated SRRM4 antibodies with alternative detection systems reveals important performance differences:

This comparison helps researchers select the optimal detection system based on specific experimental requirements, available equipment, and desired outcomes.

What experimental design would best address contradictory findings regarding SRRM4 expression in different neural subtypes?

To resolve contradictory findings regarding SRRM4 expression patterns in neural subtypes, implement this comprehensive experimental design:

• Multi-method validation approach:

  • Compare results from HRP-conjugated antibody detection with RNA-seq, qPCR, and in situ hybridization

  • Use multiple antibodies targeting different SRRM4 epitopes

  • Employ both polyclonal and monoclonal antibodies to balance sensitivity and specificity

• Carefully defined neural populations:

  • Use well-characterized cell sorting methods to isolate specific neural subtypes

  • Implement single-cell approaches to resolve heterogeneity within populations

  • Correlate with established cell type-specific markers for precise identification

• Developmental timeline consideration:

  • Examine expression at precisely defined developmental stages

  • Include multiple time points to capture dynamic expression changes

  • Standardize age/stage definitions across experiments

• Cross-species validation:

  • Compare expression patterns across species with well-conserved neural development

  • Use antibodies validated for cross-reactivity with target species

  • Identify evolutionarily conserved expression patterns that may reflect core functions

• Quantitative analysis framework:

  • Implement rigorous quantification methods with standardized thresholds

  • Use automated image analysis to eliminate observer bias

  • Apply appropriate statistical tests with corrections for multiple comparisons

What are the most promising future research directions for SRRM4 that would benefit from optimized antibody detection methods?

Several promising research directions for SRRM4 would benefit from optimized antibody detection:

• Neurodevelopmental disorder investigations:

  • Map SRRM4 expression in neurodevelopmental disorder models

  • Correlate splicing aberrations with SRRM4 expression levels in autism, intellectual disability, and related conditions

  • Develop high-throughput screening assays for compounds that normalize SRRM4-dependent splicing

• Neurodegenerative disease connections:

  • Examine SRRM4-mediated alternative splicing in age-related neurodegenerative conditions

  • Investigate potential links between SRRM4 dysfunction and protein aggregation

  • Explore therapeutic strategies targeting SRRM4-regulated splicing events

• Cancer biology applications:

  • Further investigate SRRM4's role in neuroendocrine differentiation across cancer types

  • Develop biomarker applications for treatment resistance prediction

  • Explore SRRM4 as a therapeutic target in neuroendocrine tumors

• Neural circuit specialization:

  • Map SRRM4 expression across functionally defined neural circuits

  • Correlate expression with electrophysiological properties and connectivity patterns

  • Investigate activity-dependent regulation of SRRM4 expression and function

• Therapeutic development:

  • Establish high-throughput screening platforms using HRP-conjugated antibodies

  • Develop reporters for SRRM4 activity to facilitate drug discovery

  • Explore RNA therapeutics targeting SRRM4-regulated splicing events

What are the key takeaways for researchers planning to use HRP-conjugated SRRM4 antibodies?

For researchers planning experiments with HRP-conjugated SRRM4 antibodies, these key considerations will maximize success:

• Carefully validate antibody specificity using multiple approaches (Western blot, peptide competition, knockout controls) before proceeding with extensive studies.

• Optimize detection protocols specifically for neural tissues, considering SRRM4's nuclear localization and neural-specific expression pattern .

• Include comprehensive controls in every experiment, particularly positive controls from neural tissues and negative controls from non-neural tissues.

• Consider the advantages and limitations of HRP conjugation compared to other detection methods, particularly when designing multiplexed experiments.

• Leverage the growing understanding of SRRM4's role in neural development and potentially in disease contexts like neuroendocrine cancer to formulate hypothesis-driven experimental designs.

• Implement rigorous quantification methods appropriate for the specific application (IHC, Western blot, ELISA) to enable reliable comparative analyses.

• Remain aware of potential cross-reactivity with related splicing factors, particularly in experimental systems with complex protein expression profiles.