SREK1 Antibody

What is SREK1 and why is it significant in molecular biology research?

SREK1 (Splicing Regulatory Glutamine/Lysine-Rich Protein 1) is a serine/arginine-rich (SR) splicing protein containing an unusual glutamic acid-lysine (EK)-rich domain. It functions as a key regulator of alternative splicing, particularly through its involvement in exon selection and SR-rich protein activity modulation. The significance of SREK1 lies in its role in RNA processing, with recent evidence showing its implications in:

• Cancer progression and prognosis, particularly in hepatocellular carcinoma (HCC)

• Potential role in obesity through interaction with SNORD115 and SNORD116

• Regulation of nonsense-mediated mRNA decay (NMD) pathways

The protein exists in different splice variants, with SREK1 L (exon 10-inclusive) and SREK1 S (exon 10-skipping) being the most studied forms, showing distinct cellular localizations and functions in disease contexts .

What are the key applications for SREK1 antibodies in research?

SREK1 antibodies serve multiple critical applications in both basic science and translational research:

• Western blotting (WB): For detection and quantification of SREK1 protein levels, typically used at dilutions of 1:500-1:2000

• Immunohistochemistry (IHC): For visualizing SREK1 protein distribution in tissue sections, particularly useful in cancer research

• Immunofluorescence: For subcellular localization studies, distinguishing between nuclear and cytoplasmic distribution patterns of SREK1 variants

• RNA immunoprecipitation (RIP): For studying SREK1-RNA interactions and identifying target transcripts

• Immunoprecipitation (IP): For investigating protein-protein interactions and identifying SREK1 binding partners

Research demonstrates that proper antibody selection is essential as the subcellular localization patterns of SREK1 variants differ significantly, with SREK1 L showing predominantly nuclear localization, while SREK1 S exhibits a more diffuse cellular distribution .

How should researchers validate SREK1 antibodies for experimental use?

Validation of SREK1 antibodies requires a comprehensive approach to ensure specificity, sensitivity, and reproducibility:

• Positive and negative controls: Validate using cell lines with known SREK1 expression levels. For negative controls, consider SREK1 knockdown (siRNA/shRNA) or knockout models

• Multiple detection methods: Cross-validate using at least two methods (e.g., WB and IHC)

• Isoform specificity testing: For studies focused on specific SREK1 variants, confirm antibody specificity for SREK1 L or SREK1 S using:

  • Overexpression systems with Flag-tagged SREK1 L and SREK1 S constructs

  • Isoform-specific knockdown controls using siRNAs targeting exon 10 (for SREK1 L) or exon 9/11 junction (for SREK1 S)

• Peptide competition assays: For antibodies raised against specific epitopes, particularly those within the EK-rich domain encoded by exon 10

• Cross-reactivity assessment: Test reactivity across species if working with non-human models, as reported reactivities include human and mouse samples

What are the optimal sample preparation protocols for SREK1 detection?

Sample preparation significantly impacts SREK1 antibody performance across different applications:

For Western Blotting:

• Use RIPA buffer supplemented with protease inhibitors and phosphatase inhibitors

• Include 1-2% SDS for complete solubilization of nuclear proteins

• Consider nuclear-cytoplasmic fractionation to assess compartment-specific expression patterns

For Immunohistochemistry:

• Formalin-fixed paraffin-embedded (FFPE) tissue sections are suitable

• Antigen retrieval using citrate buffer (pH 6.0) is typically effective

• SREK1 L shows predominantly nuclear staining in tumor tissues with weaker cytoplasmic signals

For RNA Immunoprecipitation:

• Crosslinking with formaldehyde (1%) stabilizes protein-RNA interactions

• Nuclear extraction protocols optimize recovery of nuclear SREK1 complexes

• RNase inhibitors are essential to preserve RNA integrity

How can SREK1 antibodies be utilized to study alternative splicing mechanisms?

SREK1 antibodies enable multifaceted approaches to investigate alternative splicing:

• Combined RIP-Seq approach:

  • Immunoprecipitate SREK1 and sequence bound RNA to identify direct splicing targets

  • This approach revealed SREK1's binding to BLOC1S5-TXNDC5 (B-T) transcript in HCC

• SREK1 variant-specific impact assessment:

  • Use SREK1 antibodies to analyze changes in alternative splicing after isoform-specific knockdown

  • RNA-seq analysis following SREK1 manipulation can reveal global splicing alterations

• Co-immunoprecipitation with splicing regulators:

  • SREK1 antibodies can co-precipitate other splicing factors like SRSF10, ELAVL1, PABPC, and MAGOH

  • This reveals regulatory complexes controlling alternative splicing events

• Sashimi plot analysis correlation:

  • Correlate SREK1 binding patterns with splice junction reads from RNA-seq

  • This approach demonstrated SRSF10's role in promoting SREK1 exon 10 inclusion

What experimental approaches effectively distinguish between SREK1 isoforms?

Distinguishing between SREK1 isoforms requires targeted strategies:

• Isoform-specific antibodies:

  • Use of antibodies targeting peptides encoded by exon 10 (e.g., CRSKEIDEKRKKDKK) specifically detects SREK1 L

  • Total SREK1 antibodies recognize both forms and can be used alongside isoform-specific antibodies for comparative analyses

• Quantitative PCR approaches:

  • Design primers spanning exon-exon junctions:

    • Exon 9-10 junction (SREK1 L-specific)

    • Exon 9-11 junction (SREK1 S-specific)

  • Calculate expressed copy numbers normalized to plasmid DNA standards

• Subcellular localization assessment:

  • Use immunofluorescence to exploit differential localization patterns:

    • SREK1 L: Predominantly nuclear

    • SREK1 S: More diffuse cellular distribution

  • This can be confirmed using Flag-tagged constructs

• Functional differentiation assays:

  • SREK1 L knockdown significantly affects cell growth and oncosphere formation

  • SREK1 S depletion shows minimal effects on growth parameters

How should SREK1 antibodies be implemented in cancer research protocols?

For effective cancer research applications, SREK1 antibody implementation should follow these protocols:

What role does SREK1 play in obesity research, and how can antibodies support this investigation?

Recent findings implicate SREK1 in obesity pathways through alternative splicing regulation:

• SNORD115/116 regulation investigations:

  • SREK1 variants in RNA recognition motif (RRM) domains impact SNORD115/116 expression

  • Use antibodies to study correlation between SREK1 variant expression and SNORD changes

• Hypothalamic neuron differentiation models:

  • Analyze SREK1 expression in iPSC-derived hypothalamic neurons

  • Compare transcriptomic profiles between wild-type and variant SREK1-expressing neurons

• Prader-Willi Syndrome (PWS) comparisons:

  • Investigate SREK1 variants associated with obesity phenotypes similar to PWS

  • Use antibodies to analyze protein expression and localization differences between variants

• Variant-specific protein interactions:

  • Compare protein interaction networks between wild-type SREK1 and obesity-associated variants

  • Identify differential binding partners that might explain phenotypic differences

How should researchers address inconsistent results when using SREK1 antibodies?

When facing inconsistent results with SREK1 antibodies, consider these methodological solutions:

• Isoform-specific expression variations:

  • Assess relative abundance of SREK1 isoforms in your model system

  • Nuclear-cytoplasmic fractionation may resolve apparent discrepancies in total protein assays

• Post-translational modifications:

  • SR proteins undergo extensive phosphorylation affecting antibody recognition

  • Include phosphatase inhibitors in lysis buffers

  • Consider using phosphorylation-specific antibodies if available

• Context-dependent expression patterns:

  • SREK1 L expression varies significantly between embryonic and adult tissues

  • Expression shows tissue-specific patterns with differences between normal and cancerous tissues

• Technical optimization matrix:

  Application	Typical Issue	Optimization Strategy
  Western Blot	Multiple bands	Nuclear extraction; Longer SDS-PAGE separation
  IHC	Weak signal	Extended antigen retrieval; Higher antibody concentration
  IF	High background	Additional blocking; Lower antibody concentration
  RIP	Poor RNA recovery	Optimize crosslinking time; Increase antibody amount

• CRISPR-based validation:

  • Generate SREK1 knockout or specific variant knock-in models

  • Use gene-edited cells as definitive controls for antibody specificity

What emerging techniques combine SREK1 antibody applications with advanced molecular analyses?

Cutting-edge research integrates SREK1 antibodies with sophisticated molecular approaches:

• CRISPR-Cas9 gene editing coupled with immunodetection:

  • Introduction of specific SREK1 variants using CRISPR-Cas9

  • HDR templates containing silent PAM site mutations prevent re-editing

  • Antibody validation of successful editing through protein expression analysis

• Integrated protein-RNA analysis platforms:

  • Combine RIP with RNA-seq (RIP-seq) to identify global SREK1 RNA targets

  • Correlate binding patterns with alternative splicing events

• Protein complex characterization:

  • SILAC proteomic strategies identify SREK1-interacting partners

  • Mass spectrometry following IP with SREK1 antibodies reveals regulatory complexes

• Sashimi plot correlation analysis:

  • Visualize RNA-seq reads across splice junctions

  • Correlate with SREK1 variant expression or binding patterns

• In vivo functional validation:

  • Adenovirus-mediated gene delivery of SREK1 variants

  • IHC detection of proliferation markers like phosphorylated Histone 3 (pH3S10)

  • Assessment of hepatocyte regeneration capacity

What emerging areas of SREK1 research will benefit from antibody-based investigations?

Several promising research directions will leverage SREK1 antibodies for new discoveries:

• Therapeutic targeting of SREK1-dependent splicing:

  • Antibody-based screening assays to identify compounds modulating SREK1 function

  • Evaluation of drug effects on SREK1 expression, localization, and interaction networks

• SREK1 in neurodevelopmental disorders:

  • Investigation of SREK1 variants in obesity and associated neurodevelopmental features

  • Analysis of SREK1's role in regulating SNORD115/116, which are implicated in Prader-Willi syndrome

• Splicing-directed therapy development:

  • Antibody-based assays to evaluate antisense oligonucleotides targeting SREK1-regulated splicing events

  • Monitoring therapy effects on SREK1 expression and splicing activity

• Single-cell protein analysis:

  • Integration of SREK1 antibodies with single-cell proteomics

  • Assessment of cell-type specific expression patterns in heterogeneous tissues

• Biomarker development for early-stage cancer detection:

  • SREK1 L/SREK1 S ratio as a potential diagnostic marker

  • Development of highly sensitive antibody-based assays for detecting SREK1 variants in liquid biopsies