spf30 Antibody

What is SPF30 and why are antibodies against it important in research?

SPF30 (Splicing Factor 30), also known as SMNDC1 (Survival Motor Neuron Domain-Containing protein 1), is an essential nuclear protein identified as a constituent of the spliceosome complex. It's a paralog of the SMN1 gene, which encodes the survival motor neuron protein associated with autosomal recessive proximal spinal muscular atrophy .

SPF30 antibodies are critical research tools because:

• They allow detection and characterization of this essential splicing factor

• They enable investigation of spliceosome assembly mechanisms

• They facilitate studies on RNA processing and its dysregulation in disease

• They support research on autoregulatory mechanisms of gene expression

SPF30 is differentially expressed across tissues, with abundant levels in skeletal muscle, and may share similar cellular functions to the SMN1 gene . Antibodies targeting SPF30 provide valuable insights into the complex machinery of pre-mRNA processing.

What are the common applications of SPF30 antibodies in molecular biology research?

SPF30 antibodies serve multiple research applications:

Common Technical Applications:

• Western blotting for protein expression analysis

• Immunoprecipitation for protein-protein interaction studies

• Immunocytochemistry for subcellular localization

• ChIP assays for investigating RNA-protein interactions

• ELISA for quantitative protein detection

Specific Research Applications:

• Investigating spliceosome assembly and function

• Studying regulation of alternative splicing

• Examining autoregulatory mechanisms of gene expression

• Exploring the relationship between splicing factors and disease

• Analyzing methylarginine-dependent protein interactions

For example, immunocytochemical staining has shown that SPF30 localizes to nuclear speckles, partly coinciding with the speckle marker SC35, indicating its role in pre-mRNA processing machinery .

How do I validate the specificity of an SPF30 antibody for my experiments?

Validation of SPF30 antibody specificity requires a multi-faceted approach:

Essential Validation Steps:

• Western blot analysis: Verify a single band at the expected molecular weight (~30 kDa)

• Positive controls: Use tissues known to express high levels of SPF30 (skeletal muscle)

• Negative controls: Use SPF30 knockout cells created via CRISPR-Cas9 as demonstrated in recent studies

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

• Orthogonal detection method: Compare results with a second antibody targeting a different epitope

Advanced Validation:

• Immunoprecipitation followed by mass spectrometry

• Testing cross-reactivity with related proteins (especially SMN1)

• Validation across multiple species if working with non-human models

Research has shown that SPF30 antibodies can successfully detect both endogenous SPF30 and tagged variants (SPF30-FLAG) in experimental settings, with the tagged version showing higher molecular weight due to the additional sequences .

What experimental conditions optimize SPF30 antibody performance in immunoprecipitation studies?

Optimizing SPF30 antibody performance for immunoprecipitation requires attention to several key parameters:

Optimal IP Conditions for SPF30:

Research indicates that SPF30 interactions with RNA and protein components can be maintained under stringent conditions of RIPA buffer , though some specific protein-protein interactions may require milder conditions.

For detecting interactions between SPF30 and methylated proteins, studies have shown that interactions between GFP-SART3 (containing SPF30 domains) and endogenous fibrillarin and coilin are methylation-dependent and can be disrupted by treatment with methyltransferase inhibitors like EPZ015666 .

How can I detect SPF30's interactions with spliceosomal components using antibody-based approaches?

Detecting SPF30's interactions with spliceosomal components requires specialized protocols:

Effective Detection Strategies:

• Co-immunoprecipitation (Co-IP):

  • Use anti-SPF30 antibody to pull down the protein complex

  • Probe with antibodies against known spliceosome components (U4/U5/U6 and U2 snRNP components)

  • Research shows SPF30 associates with both U4/U5/U6 and U2 snRNP components

• Proximity Ligation Assay (PLA):

  • Combines antibody recognition with DNA amplification for in situ detection

  • Requires antibodies from different species (anti-SPF30 and anti-spliceosomal component)

  • Provides spatial resolution of interactions within the nucleus

• Mass Spectrometry Analysis after IP:

  • Studies show recombinant GST-SPF30 fusion protein associates with complexes containing core Sm and U4/U5/U6 tri-snRNP proteins

  • Strong association with U4/U6-90 has been demonstrated

• Far-Western Analysis:

  • Can detect direct protein-protein interactions

  • Use recombinant SPF30 protein as a probe against spliceosomal components

  • Research has employed GST-SART3(HAT) recombinant protein containing SPF30 domains to probe cell lysates successfully

The choice of method depends on whether you're investigating direct or indirect interactions, complex stability, and the cellular context of the interaction.

What controls should I include when studying SPF30's autoregulatory mechanisms using antibodies?

Studying SPF30's autoregulatory mechanisms requires rigorous controls:

Essential Controls for Autoregulation Studies:

• Expression Level Controls:

  • Titrated expression of SPF30 (endogenous vs. overexpressed)

  • Quantitative western blot analysis to monitor protein levels

  • Research shows SPF30-FLAG expression reduced endogenous SPF30 expression by up to 60% after 48h compared to uninduced cells

• Splicing Pattern Controls:

  • RT-PCR analysis of SPF30 mRNA variants (with/without cassette exon, with/without exon 4a)

  • Primers specific for different splice variants

  • Studies demonstrate SPF30 overexpression increases inclusion of cassette exon and exon 4a

• NMD Inhibition Control:

  • Treatment with cycloheximide (CHX) to inhibit nonsense-mediated mRNA decay

  • RT-PCR analysis before and after CHX treatment

  • Research shows CHX treatment increases detection of SPF30 transcripts containing exon 4a

• Domain Function Controls:

  • Expression of SPF30 deletion mutants (e.g., ΔC5 deletion mutant Δ218–238)

  • Analysis of RNA-binding properties using EMSA

  • Studies indicate the C-terminal region is critical for autoregulation

A comprehensive experimental design includes both positive controls (known SPF30 splice variants) and negative controls (non-targeting siRNAs, unrelated protein overexpression) to ensure specificity of the observed autoregulatory effects.

How can I distinguish between the functions of SPF30 and related proteins like SMN1 using antibody-based approaches?

Distinguishing between SPF30 and SMN1 functions requires sophisticated antibody-based strategies:

Differential Analysis Approaches:

• Sequential Immunodepletion:

  • Deplete cell extracts of SPF30 using specific antibodies

  • Test for spliceosome assembly and function

  • Perform reciprocal experiment with SMN1 depletion

  • Studies show that in the absence of SPF30, the preformed tri-snRNP fails to assemble into the spliceosome

• Domain-Specific Antibodies:

  • Generate antibodies against unique domains not conserved between SPF30 and SMN1

  • Use for immunoprecipitation followed by functional assays

  • Target the C-terminal region of SPF30, which is responsible for its autoregulation

• Structured Functional Rescue Experiments:

  • Deplete endogenous protein using siRNA targeting untranslated regions

  • Rescue with wild-type or mutated versions of SPF30 or SMN1

  • Analyze spliceosome assembly and RNA processing

  • Research demonstrates that SPF30 knockdown affects specific splicing events that can be rescued by wild-type but not mutant SPF30

• Methylarginine-Dependent Interaction Analysis:

  • Compare binding partners under conditions that modify arginine methylation

  • Use methyl-specific antibodies to confirm modification status

  • Studies show SPF30 binds methylarginine-marked motifs with specificity different from SMN1

The key is to exploit the functional and structural differences between these paralogous proteins while controlling for their overlapping activities.

What are the best methodological approaches for investigating the role of SPF30 in RNA splicing regulation?

Investigating SPF30's role in RNA splicing regulation requires integrated methodological approaches:

Comprehensive Experimental Strategy:

• Splicing-Specific RNA-Seq Analysis:

  • Perform SPF30 knockdown or overexpression

  • Analyze alternative splicing events globally

  • Focus on exon inclusion/exclusion, alternative 5' and 3' splice sites

  • Research shows SPF30 affects inclusion of cassette exon and exon 4a in its own transcript

• In Vivo Splicing Assays:

  • Design minigenes containing SPF30 target sequences

  • Introduce mutations in potential regulatory elements

  • Measure splicing outcomes using reporters like NanoLuc Luciferase

  • Studies have used this approach to identify sequences required for exon 4a inclusion

• RNA-Protein Binding Analysis:

  • RNA immunoprecipitation (RIP) using SPF30 antibodies

  • Crosslinking and immunoprecipitation (CLIP) for direct binding sites

  • In vitro binding assays with synthetic RNA oligonucleotides

  • Research demonstrates SPF30 binds directly to specific sequences within exon 4

• Structure-Function Analysis:

  • Create domain deletion mutants (e.g., ΔC1–ΔC5)

  • Assess RNA binding using EMSA and protein-RNA docking simulations

  • Evaluate splicing activity in cellular contexts

  • Studies show both C-terminal α-helix and kink-like structure of SPF30 are required for RNA binding

Results from these approaches can be integrated to build a comprehensive model of SPF30's role in splicing regulation, from direct RNA recognition to effects on spliceosome assembly.

How can discrepancies in SPF30 antibody performance across different experimental systems be reconciled?

Reconciling discrepancies in SPF30 antibody performance requires systematic troubleshooting and understanding of potential confounding factors:

Systematic Reconciliation Approach:

• Epitope Accessibility Analysis:

  • Map the epitope recognized by the antibody

  • Assess potential masking by protein-protein interactions

  • Evaluate post-translational modifications affecting recognition

  • Different fixation methods for immunocytochemistry can dramatically affect epitope accessibility

• Isoform-Specific Recognition:

  • SPF30 undergoes alternative splicing producing variants with cassette exon and exon 4a

  • Verify whether antibody epitopes span exon junctions affected by splicing

  • Design experiments to detect all relevant isoforms

  • Consider targeted mass spectrometry to identify all protein variants present

• Cross-Species Reactivity Assessment:

  • Perform sequence alignment of SPF30 across species of interest

  • Evaluate conservation of epitope regions

  • Test antibody performance in multiple species systematically

  • Research shows conservation differences, with SPF30 HAT regions from vertebrate species binding to coilin-SDMA peptide, but not from C. elegans

• Experimental Condition Matrix:

Addressing these variables systematically can help reconcile discrepancies and establish reliable protocols for SPF30 antibody use across experimental systems.

How can SPF30 antibodies be employed to investigate the protein's role in methylarginine-marked motif recognition?

Investigating SPF30's role in methylarginine-marked motif recognition requires specialized antibody applications:

Advanced Methodological Approaches:

• Methylation-Specific Co-Immunoprecipitation:

  • Use SPF30 antibodies to pull down protein complexes

  • Probe with antibodies specific for symmetrically dimethylated arginine (SDMA)

  • Compare samples with and without methyltransferase inhibitors

  • Research shows inhibition of PRMT5 with EPZ015666 reduces the interaction between GFP-SART3 (containing SPF30 domains) and endogenous fibrillarin and coilin

• In Vitro Binding Assays with Methylated Peptides:

  • Generate synthetic peptides with defined methylation patterns

  • Use GST-SART3(HAT) recombinant protein containing SPF30 domains

  • Assess binding using pull-down assays or isothermal titration calorimetry

  • Studies determined that GST-SART3 HAT repeats bind coilin SDMA peptides with an equilibrium dissociation constant (KD) of 15–18 μM

• Far-Western Analysis with Methylation Controls:

  • Prepare cell lysates with varying methylation states

  • Probe with recombinant SPF30 protein

  • Compare binding patterns between normal and AdOx-treated extracts

  • Research shows GST-SART3(HAT) signal was enriched on wild-type methylated extracts but not AdOx-treated extracts

• Structure-Guided Mutagenesis Combined with Binding Studies:

  • Create SPF30 mutants at key aromatic residues (Y112A, F142A, Y180A, and W377A)

  • Test interaction with known methylarginine-containing proteins

  • Perform IP experiments in knockout/rescue systems

  • Studies demonstrate that these mutants have reduced ability to co-precipitate methylated binding partners

These approaches provide a comprehensive framework for dissecting SPF30's role in reading methylarginine marks and its functional significance in cellular contexts.

What are the most effective strategies for using SPF30 antibodies in transmission-blocking vaccine research?

This question appears to reflect a confusion between two different uses of "SPF30" in the research literature. In vaccine research, particularly for malaria, "Pfs230" (not SPF30) is a transmission-blocking vaccine antigen. SPF30 is a splicing factor unrelated to vaccine development. To clarify this distinction:

Correction and Clarification:

SPF30 (Splicing Factor 30) and Pfs230 (Plasmodium falciparum surface protein 230) are entirely different proteins:

• SPF30/SMNDC1: A human splicing factor involved in spliceosome assembly and RNA processing

• Pfs230: A malaria parasite protein that is a target for transmission-blocking vaccines

The search results contain information about both proteins, which may have caused confusion. While antibody research is relevant to both, their applications are fundamentally different:

• SPF30 antibodies: Used to study RNA splicing and gene expression regulation

• Anti-Pfs230 antibodies: Used in malaria transmission-blocking vaccine development

For researchers interested in malaria transmission-blocking vaccines, the relevant information concerns Pfs230, which is a distinct topic from SPF30 antibodies in splicing research.

How do experimental conditions affect the detection of SPF30's autoregulatory splicing mechanisms?

Detection of SPF30's autoregulatory splicing mechanisms is highly sensitive to experimental conditions:

Critical Experimental Parameters:

• NMD Inhibition Timing and Concentration:

  • Cycloheximide (CHX) treatment is essential to prevent degradation of alternatively spliced transcripts

  • Optimal conditions: 100 μg/mL CHX for 4-6 hours prior to RNA extraction

  • Deep sequencing after CHX treatment reveals that SPF30 overexpression increases the percentage of exon 4a inclusion from 26.0% to 54.7% in transcripts with cassette exon

• SPF30 Expression Level Controls:

  • Dosage effects significantly impact splicing patterns

  • Inducible expression systems allow controlled titration

  • Western blot analysis showed that SPF30-FLAG expression plateaued at 48h after induction with doxycycline

• RNA Extraction Timing:

  • Early extraction may miss secondary effects on splicing

  • Time-course experiments capture the dynamic nature of autoregulation

  • RT-qPCR with specific primers for different splicing events provides quantitative assessment

• Primer Design for Splicing Detection:

  • Critical for accurate quantification of splice variants

  • Primers spanning specific exon junctions

  • RT-PCR using specific primers for exon 4a revealed increased abundance after CHX treatment

• Cell Type Considerations:

  • Different cell lines may have varying basal splicing machinery

  • Endogenous SPF30 levels affect experimental outcomes

  • Controlled experiments using knockout/rescue approaches provide cleaner results

A comprehensive experimental design accounts for these variables and includes controls for RNA quality, splicing efficiency, and potential off-target effects of experimental manipulations.

What methodological advances enable investigation of the structural basis for SPF30's RNA binding specificity?

Recent methodological advances provide powerful tools for investigating SPF30's RNA binding specificity:

Cutting-Edge Structural Biology Approaches:

• Integrated Structural Proteomics:

  • Combine hydrogen-deuterium exchange mass spectrometry (HDX-MS) with crosslinking-MS

  • Map SPF30 protein-RNA interaction surfaces

  • Identify conformational changes upon RNA binding

  • Research demonstrates that both the C-terminal α-helix and kink-like structure of SPF30 are required for RNA binding

• In Silico Protein-RNA Docking with Experimental Validation:

  • Generate computational models of SPF30-RNA complexes

  • Validate with mutagenesis and binding assays

  • Docking simulations with SPF30 mutants reveal different binding patterns with transcripts containing exon 4a versus wild-type SPF30

  • Models can reveal how SPF30 binds to junctions of introns 3 and 4a

• Domain-Specific Analysis through Mutant Series:

  • Create deletion series (ΔC1-ΔC5) targeting structural elements

  • Assess RNA binding through EMSA and functional assays

  • Research shows the ΔC5 deletion mutant (Δ218–238) retains RNA-binding properties while losing autoregulatory function

• RNA Structure Probing Combined with Protein Footprinting:

  • Use SHAPE (Selective 2′-hydroxyl acylation analyzed by primer extension) to determine RNA structure

  • Map protein binding sites through protection assays

  • Identify structural changes in RNA upon protein binding

• Single-Molecule Approaches:

  • Fluorescence techniques to visualize individual binding events

  • Measure binding kinetics and conformational dynamics

  • Determine the order of assembly and binding cooperativity

The integration of these approaches provides a comprehensive view of how SPF30's structure determines its specificity for particular RNA sequences and how this relates to its function in splicing regulation.

What emerging technologies will advance our understanding of SPF30 function using antibody-based approaches?

Several emerging technologies promise to revolutionize SPF30 research through advanced antibody applications:

Frontier Methodologies:

• Proximity-Dependent Labeling Technologies:

  • APEX2 or BioID fusions with SPF30 combined with specific antibodies

  • Map the dynamic SPF30 interactome in living cells

  • Identify transient interactions during spliceosome assembly

  • Can be combined with temporal control for process-specific interaction mapping

• Super-Resolution Microscopy with Antibody-Based Detection:

  • STORM/PALM imaging of SPF30 within nuclear speckles

  • Multi-color imaging to resolve spatial relationships with other spliceosomal components

  • Track dynamic assembly/disassembly processes at nanometer resolution

  • Research has shown SPF30 localizes to nuclear speckles, partly coinciding with the speckle marker SC35

• Antibody-Enabled Spatial Transcriptomics:

  • Combine SPF30 immunostaining with in situ sequencing

  • Map splicing regulation events at subcellular resolution

  • Identify localized regulation of RNA processing

• CRISPR-Based Tagging for Endogenous Antibody Recognition:

  • Insert small epitope tags at the endogenous SPF30 locus

  • Preserve native expression patterns and regulation

  • Enable clean immunoprecipitation without overexpression artifacts

  • Research demonstrates successful knockout of SART3 in HeLa cells using CRISPR-Cas9 before reintroducing tagged versions

• Nanobody Development for Intracellular Targeting:

  • Engineer small, single-domain antibodies against SPF30

  • Express inside living cells to track or perturb function

  • Combine with optogenetic tools for temporal control