SRRM4 Antibody, Biotin conjugated

What is SRRM4 and what cellular functions does it perform?

SRRM4 (serine/arginine repetitive matrix 4) is a nuclear protein with a canonical length of 611 amino acid residues and molecular weight of approximately 68.6 kDa in humans. It functions primarily as a neural-specific splicing factor that promotes alternative splicing and inclusion of neural-specific exons in target mRNAs . SRRM4 belongs to the NSR100 protein family and is involved in critical cellular processes including neuronal differentiation and specialized mRNA processing . The protein is predominantly expressed in neuronal cells, where it plays a crucial role in regulating neuron-specific splicing events that contribute to proper nervous system development .

How does SRRM4 differ from other splicing factors in the same family?

SRRM4 shares functional similarities with SRRM3, another neuronal splicing regulator with SRRM4-like activity. Both proteins enhance the incorporation of alternative exons into mRNAs through similar mechanisms, with SRRM4 showing slightly higher activity compared to SRRM3 in experimental models . This activity difference correlates with their relative expression levels in cells .

Unlike general splicing factors like SRSF1, which had no effect on the incorporation of alternative exons in experimental settings, SRRM3 and SRRM4 specifically recognize intronic UGC motifs necessary for neural-specific alternative splicing . Their expression patterns also differ: SRRM3 shows weak signal in Purkinje cells but strong expression in cells of the granular and molecular layers of the cerebellum, creating differential splicing patterns across neuronal subtypes .

What are the established applications for biotin-conjugated SRRM4 antibodies?

Biotin-conjugated SRRM4 antibodies are primarily utilized in ELISA (Enzyme-Linked Immunosorbent Assay) applications according to supplier information . The biotin conjugation provides several advantages in detection systems, particularly when combined with streptavidin-based visualization methods. While unconjugated SRRM4 antibodies have broader application ranges including Western Blot (WB), Immunohistochemistry (IHC), and Immunofluorescence, the biotin-conjugated version offers enhanced sensitivity specifically optimized for ELISA-based detection systems .

What are the optimal sample preparation procedures when using SRRM4 antibodies?

For optimal results with SRRM4 antibodies, sample preparation should account for the nuclear localization of the target protein. Based on research applications:

• For cell/tissue lysates: Nuclear extraction protocols are recommended since SRRM4 is predominantly localized in the nucleus . Phosphatase inhibitors should be included in extraction buffers as SRRM4 undergoes phosphorylation as a post-translational modification .

• For tissue sections: Paraformaldehyde fixation (typically 10%) followed by paraffin embedding has been successfully used in studies examining SRRM4 expression in xenograft models . This preparation method preserves protein structure while maintaining tissue architecture for localization studies.

• For neuronal tissues: Special attention should be given to preserving RNA-protein interactions if studying SRRM4's role in alternative splicing, potentially using crosslinking techniques prior to immunoprecipitation .

How can researchers validate the specificity of SRRM4 antibodies?

Validating SRRM4 antibody specificity requires multiple complementary approaches:

• Peptide competition assays: Using the immunizing peptide (such as aa 400-479 for certain antibodies) to block antibody binding and confirm signal specificity .

• Positive and negative control tissues: Comparing tissues with known high expression (neuronal cells) versus low expression, and correlating with mRNA expression data .

• Knockdown/overexpression controls: Comparing signal in SRRM4 overexpression models (via lentiviral transduction as used in multiple studies) versus control vector transduced cells .

• Cross-species validation: Testing reactivity across species with consideration of sequence homology percentages (Cow: 86%; Dog: 93%; Guinea Pig: 93%; Horse: 93%; Human: 100%; Mouse: 100%; Rabbit: 92%; Rat: 100%) .

What are the advantages of biotin-conjugated SRRM4 antibodies over unconjugated versions?

Biotin-conjugated SRRM4 antibodies offer several methodological advantages:

• Enhanced sensitivity: The biotin-streptavidin interaction is one of the strongest non-covalent biological interactions (Kd ≈ 10^-15 M), providing signal amplification opportunities.

• Versatile detection options: Compatible with multiple visualization systems including streptavidin-HRP, streptavidin-fluorophores, or streptavidin-gold particles.

• Reduced background in certain applications: Particularly valuable in ELISA applications where the biotin-conjugated antibody serves as a detection antibody in sandwich assays.

• Compatibility with multi-labeling experiments: Biotin-conjugated antibodies can be used alongside other directly labeled antibodies when detecting multiple targets simultaneously.

How can SRRM4 antibodies be used to investigate neuroendocrine differentiation in cancer models?

SRRM4 antibodies serve as valuable tools in studying neuroendocrine differentiation in cancer, particularly in prostate cancer progression to neuroendocrine prostate cancer (NEPC):

• Lentiviral-based models: Researchers have developed multiple cell models by transducing prostate cancer cells (including LNCaP, 22Rv1, PC-3, and DU145) with lentiviral vectors encoding SRRM4. Antibodies can detect successful expression of SRRM4 in these models via immunoblotting .

• Xenograft studies: SRRM4 antibodies have been used in immunohistochemistry of xenograft sections to assess SRRM4 expression and correlate it with neuroendocrine markers like synaptophysin (SYP) .

• Mechanistic investigations: Antibodies enable the study of how SRRM4 activates pluripotency gene networks, including SOX2. This SRRM4-SOX2 axis is present in a subset of NEPC patient cohorts, patient-derived xenografts, and clinically relevant transgenic mouse models .

• Differential diagnosis: SRRM4 antibody staining could potentially differentiate between adenocarcinoma and neuroendocrine tumors based on SRRM4 expression patterns and associated splicing signatures.

What approaches can assess SRRM4's interaction with target pre-mRNAs?

Investigating SRRM4's interaction with target pre-mRNAs requires specialized techniques where SRRM4 antibodies play essential roles:

• RNA immunoprecipitation (RIP): SRRM4 antibodies can immunoprecipitate the protein along with bound RNAs, which can then be analyzed by RT-PCR or sequencing to identify bound targets.

• CLIP-seq (Cross-linking immunoprecipitation followed by sequencing): This technique allows mapping of precise RNA-binding sites of SRRM4 using UV crosslinking followed by immunoprecipitation with SRRM4 antibodies.

• Minigene splicing assays: As demonstrated in research, SRRM4 affects splicing of minigene constructs containing approximately 800-bp genomic regions with SRRM4-regulated exons and neighboring intronic sequences . Antibodies can confirm SRRM4 expression in these experimental systems.

• Splicing reporter systems: SRRM4 antibodies can validate expression levels when studying SRRM4's effect on reporter constructs containing UGC motifs, which research has shown are necessary for SRRM4-dependent splicing .

How do SRRM3 and SRRM4 activities overlap in neuronal systems?

Research using antibodies against SRRM3 and SRRM4 has revealed significant functional overlap between these factors:

• Compensatory mechanisms: Studies in mouse models show that SRRM4 limits the severity of splicing defects in SRRM3-deficient (gt/gt Srrm3) mice, indicating functional redundancy .

• Tissue-specific differences: SRRM3 deficiency causes more severe splicing defects in the Purkinje layer than in the granular layer of the cerebellum, suggesting differential compensation by SRRM4 across neural cell types .

• Double mutant studies: Analysis of mice with mutations in both Srrm3 and Srrm4 (dMut mice) revealed that incorporation of target exons into mRNA was reduced in the neocortex much more severely than in single-gene mutant mice, providing strong evidence for overlapping functions .

• Target specificity: Both factors redundantly regulate alternative splicing of exon 4 of Rest in the neocortex, supporting molecular function overlap .

What are common sources of background when using biotin-conjugated antibodies?

When working with biotin-conjugated SRRM4 antibodies, several factors can contribute to background issues:

• Endogenous biotin interference: Tissues with high endogenous biotin (like brain, kidney, and liver) can cause background when using avidin/streptavidin detection systems.

• Non-specific binding: Secondary reagents (streptavidin conjugates) may bind non-specifically to hydrophobic regions of certain tissues.

• Fc receptor binding: Tissues containing cells with Fc receptors (like immune cells) can bind the Fc region of antibodies non-specifically.

Mitigation strategies:

• Pre-block tissues with avidin/biotin blocking kits before applying biotin-conjugated antibodies

• Use proper blocking reagents (BSA, normal serum, or commercial blockers)

• Include isotype control antibodies conjugated to biotin

• Optimize antibody concentrations through titration experiments

How can researchers address contradictory findings when studying SRRM4 across different cell types?

When researchers encounter contradictory findings with SRRM4 antibodies across different cell types, systematic troubleshooting approaches include:

• Cell-type specific splicing programs: SRRM4 induces heterogeneous transcriptomes and phenotypes among different cell models tested . This natural variability should be considered when interpreting apparently contradictory results.

• Expression level validation: Confirm SRRM4 expression levels using multiple methods (qPCR, immunoblotting, immunohistochemistry) as different detection thresholds might explain discrepancies.

• Co-factor dependencies: SRRM4 function may depend on co-factors that vary between cell types. For example, in DU145 cells, SRRM4 activates a pluripotency gene network including SOX2, which isn't observed in all cell types .

• Tissue context considerations: When working with tissue samples, consider the heterogeneous nature of tissues. Laser capture microdissection (as used in studies of Purkinje and granular layers) may help isolate specific cell populations for more accurate analysis .

What controls are essential when studying differential splicing patterns using SRRM4 antibodies?

When investigating differential splicing patterns using SRRM4 antibodies, researchers should implement these essential controls:

• Expression validation controls:

  • Positive control: Tissues known to express SRRM4 (neuronal tissues)

  • Negative control: Tissues with minimal SRRM4 expression

• Splicing validation controls:

  • RT-PCR analysis of known SRRM4-regulated exons (like exon 4 of Rest)

  • Minigene assays with and without SRRM4 expression

• Specificity controls:

  • Comparison with related splicing factors (SRRM3 or heterologous SRSF1)

  • Antibody validation using SRRM4 overexpression and knockdown models

• Functional redundancy controls:

  • Assessment of redundant splicing factors (like SRRM3) that may compensate for SRRM4 function

  • Analysis of UGC motifs necessary for SRRM4-dependent splicing

What sequence information is important when selecting SRRM4 antibodies?

Selection of SRRM4 antibodies should consider epitope location and sequence conservation across species:

Table 1: Sequence Homology of Human SRRM4 Across Species

Key peptide sequences used for antibody generation include:

• aa 270-319 region (used for certain polyclonal antibodies)

• aa 400-479 region (used for biotin-conjugated antibodies)

• "KTASPLTTSR GRSQEYDSGN DTSSPPSTQT SSARSRGQEK GSPSGGLSKS" sequence

When selecting antibodies for cross-species applications, the high homology between human, mouse, and rat SRRM4 (100%) makes most anti-human antibodies suitable for rodent studies .

How do SRRM4 antibody applications compare across different experimental platforms?

Table 2: Comparison of SRRM4 Antibody Applications and Platforms

✓✓✓ = Highly recommended; ✓✓ = Recommended; ✓ = Applicable but not optimal

The data demonstrates that while biotin-conjugated SRRM4 antibodies excel in ELISA applications, unconjugated versions offer greater versatility across multiple experimental platforms .

What molecular mechanisms link SRRM4 to neuroendocrine differentiation in cancer?

Research using SRRM4 antibodies has helped elucidate the molecular mechanisms by which SRRM4 contributes to neuroendocrine differentiation in cancer:

• Global splicing program: SRRM4 induces a global NEPC-specific RNA splicing signature in prostate cancer cell models. This program is detectable using antibodies to confirm SRRM4 expression alongside splicing changes .

• SOX2 activation pathway: SRRM4 enhances SOX2 expression in both time- and dose-dependent manners in DU145 cells. This SRRM4-SOX2 axis is present in subsets of NEPC patient cohorts, patient-derived xenografts, and transgenic mouse models .

• Pluripotency network activation: SRRM4 drives NEPC progression via induction of a pluripotency gene network, with SOX2 playing a critical role in this process .

• Phenotypic consequences: SRRM4 expression leads to:

  • Neuronal morphological changes

  • Accelerated cell proliferation

  • Enhanced resistance to apoptosis

  • Development of xenografts with classic NEPC phenotypes

The mechanisms identified highlight SRRM4's role as a key driver of lineage plasticity in prostate cancer progression to neuroendocrine phenotypes, with distinct pathways compared to other models of neuroendocrine differentiation .

What emerging applications of SRRM4 antibodies show the most promise?

Emerging applications of SRRM4 antibodies that show significant research potential include:

• Biomarker development: SRRM4 antibodies could serve as diagnostic or prognostic tools for identifying neuroendocrine differentiation in prostate and potentially other cancers .

• Therapeutic target assessment: As SRRM4 drives NEPC progression, antibodies can help evaluate the efficacy of interventions targeting SRRM4 or its downstream pathways.

• Developmental neurobiology: SRRM4 antibodies can elucidate the temporal and spatial expression patterns during neural development and differentiation .

• Comparative neuroscience: The high conservation of SRRM4 across species enables cross-species studies of neuronal splicing regulation .

• Single-cell approaches: Combining SRRM4 antibodies with single-cell technologies could reveal cell-type specific splicing programs in heterogeneous tissues like brain or tumors.