SCAF8 Antibody

What is SCAF8 and what is its primary function in cellular transcription?

SCAF8 (SR-Related CTD Associated Factor 8, also known as RBM16) is a protein involved in mRNA splicing and gene expression regulation. Its primary function is as an anti-terminator protein that prevents early mRNA termination during transcription. Specifically, SCAF8 works redundantly with SCAF4 to suppress the use of early, alternative polyadenylation (polyA) sites, thereby preventing the accumulation of non-functional truncated proteins .

Additionally, independent of its anti-termination role, SCAF8 functions as a positive regulator of transcript elongation. SCAF8 mechanistically associates with the phosphorylated C-terminal heptapeptide repeat domain (CTD) of the largest RNA polymerase II subunit (POLR2A) and subsequently binds nascent RNA upstream of early polyadenylation sites to prevent premature mRNA transcript cleavage and polyadenylation .

What are the technical specifications of currently available SCAF8 antibodies?

Several research-grade SCAF8 antibodies are currently available, with varying specifications:

Most commercially available antibodies are polyclonal, produced in rabbits, and optimized for Western blot applications. The calculated molecular weight of SCAF8 is 141 kDa, though it's observed at approximately 170 kDa in Western blot analyses, likely due to post-translational modifications .

How should I optimize Western blot protocols specifically for SCAF8 detection?

For optimal SCAF8 detection using Western blot, I recommend the following methodology:

• Sample preparation: Extract nuclear proteins as SCAF8 is exclusively nuclear-localized . Use a lysis buffer containing phosphatase inhibitors to preserve any phosphorylated forms.

• Gel selection: Use 6-8% SDS-PAGE gels to properly resolve the high molecular weight SCAF8 protein (observed at ~170 kDa).

• Transfer conditions: Perform wet transfer at low voltage (30V) overnight at 4°C to ensure complete transfer of this large protein.

• Blocking: Use 5% non-fat dry milk in TBST for 1 hour at room temperature.

• Primary antibody: Dilute SCAF8 antibodies at 1:500 to 1:1000 in blocking buffer and incubate overnight at 4°C .

• Controls: Include positive controls such as HeLa cell lysate, mouse brain, or rat brain lysates, which have shown reliable SCAF8 expression .

• Detection method: Use chemiluminescence for high sensitivity detection.

The observed molecular weight of approximately 170 kDa (rather than the calculated 141 kDa) should be expected and is consistent with published literature .

What immunoprecipitation protocols work best for studying SCAF8 interactions with RNA polymerase II?

Based on published research methodologies, the following IP protocol is recommended for studying SCAF8-RNAPII interactions:

• Crosslinking: Perform dual crosslinking with DSG (disuccinimidyl glutarate) followed by formaldehyde to capture transient protein-protein interactions.

• Lysis conditions: Use a nuclear extraction buffer containing 20 mM HEPES pH 7.9, 150 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5% NP-40, and 10% glycerol supplemented with protease and phosphatase inhibitors.

• Pre-clearing: Pre-clear lysates with protein A/G beads for 1 hour at 4°C.

• Antibody binding: Incubate pre-cleared lysates with 2-5 μg of SCAF8 antibody overnight at 4°C.

• Bead capture: Add protein A/G beads and incubate for 2-3 hours at 4°C.

• Washing: Perform stringent washes with increasing salt concentrations (150-300 mM NaCl).

• Detection: Probe Western blots with antibodies against different phosphorylated forms of RNAPII CTD (Ser2P, Ser5P, Ser7P, Thr4P, and Tyr1P) .

This approach has successfully demonstrated that SCAF8 associates specifically with the transcriptionally engaged, hyper-phosphorylated form of RNAPII, with a strong preference for RNAPII CTD phosphorylated on both Ser2 and Ser5 .

How can SCAF8 antibodies be employed in ChIP-seq experiments to study genome-wide binding patterns?

For effective ChIP-seq experiments to study SCAF8 genome-wide binding patterns:

• Crosslinking optimization: Use dual crosslinking with 2 mM DSG for 45 minutes followed by 1% formaldehyde for 10 minutes to capture both direct DNA interactions and indirect associations through the RNAPII complex.

• Sonication conditions: Optimize sonication to obtain chromatin fragments of 200-300 bp.

• Antibody selection: Use antibodies validated for immunoprecipitation, preferably those targeting epitopes outside the CTD-interaction domain (CID) to avoid competing with RNAPII binding.

• Controls: Include:

  • Input controls

  • IgG negative controls

  • RNAPII ChIP-seq in parallel for correlation analysis

  • SCAF4 ChIP-seq for comparative binding analysis

• Analysis approach: Focus analysis on:

  • Enrichment near alternative polyadenylation sites

  • Co-occupancy with RNAPII (especially Ser2/5 phosphorylated forms)

  • Proximity to splice junctions (SCAF8 binding clusters are significantly enriched near splice sites)

Research has shown that SCAF8 binding sites significantly overlap with SCAF4 (approximately 65% overlap), and these sites are enriched near RNA processing regions, particularly near alternative polyadenylation sites and splice junctions .

What methods can distinguish between SCAF8 and SCAF4 functions despite their functional redundancy?

Despite their functional redundancy in preventing early mRNA termination, SCAF8 and SCAF4 have distinct individual functions that can be distinguished using the following methodological approaches:

• Single knockout studies: Generate separate SCAF4 and SCAF8 knockout cell lines using CRISPR-Cas9, as published studies have demonstrated these single knockouts are viable while the double knockout is lethal .

• RNA-seq analysis of single KOs: Compare transcriptome profiles between:

  • Wild-type cells

  • SCAF4 KO cells

  • SCAF8 KO cells

  • SCAF4/8 double KO cells with doxycycline-inducible rescue constructs

• Nascent RNA labeling: Use 4-thiouridine (4SU) pulse labeling followed by sequencing (TT-seq) to detect changes in nascent transcription, particularly transcriptional readthrough events .

• Key phenotypes to monitor:

  • SCAF8 KO: Monitor for changes in transcript elongation rates

  • SCAF4 KO: Analyze for transcriptional readthrough beyond normal termination sites (increased nascent RNA in the 50 kb region downstream of the transcription end site)

  • Double KO: Assess alternative last exon (ALE) usage and alternative polyadenylation site selection

Research has revealed that SCAF8 functions independently as an elongation factor, while SCAF4 is required for correct termination at canonical, distal transcription termination sites when SCAF8 is present .

Why might Western blots show unexpected band sizes when detecting SCAF8?

When Western blots show unexpected band sizes for SCAF8, consider the following technical explanations and solutions:

• Higher molecular weight than predicted: SCAF8's calculated MW is 141kDa, but it typically appears at ~170kDa on Western blots due to:

  • Post-translational modifications, particularly phosphorylation

  • The high proline content affecting protein migration

  • Solution: Use positive controls like HeLa cell lysate where SCAF8 is well-characterized

• Multiple bands: These may represent:

  • Different phosphorylation states

  • Alternative splice variants

  • Proteolytic degradation

  • Solution: Include phosphatase treatment controls and use fresh samples with complete protease inhibitors

• Lower molecular weight bands only: May indicate:

  • Proteolytic degradation during sample preparation

  • Expression of truncated forms in certain conditions

  • Solution: Use stringent lysis conditions with multiple protease inhibitors and keep samples cold throughout processing

• No band detection: Consider:

  • Expression levels vary significantly by tissue type (highest in ovary and uterus)

  • Solution: Use tissues known to express SCAF8 at higher levels, such as brain, testis, or HeLa cells

How can I validate SCAF8 antibody specificity for my particular experimental system?

A comprehensive antibody validation strategy for SCAF8 should include:

• Genetic models:

  • Use CRISPR/Cas9 SCAF8 knockout cells as negative controls

  • For added stringency, use conditional knockouts with doxycycline-inducible rescue constructs as described in published literature

• Epitope competition:

  • Pre-incubate antibody with excess immunizing peptide (if available)

  • Verify signal reduction in Western blot or immunostaining

• Cross-reactivity assessment:

  • Test antibody against both SCAF4 and SCAF8 recombinant proteins to confirm specificity, as these proteins share 38% identity and 50% similarity

• Orthogonal methods:

  • Compare results with different antibodies targeting distinct SCAF8 epitopes

  • Correlate protein detection with mRNA expression data

• Tagged protein controls:

  • Express tagged SCAF8 (e.g., GFP-SCAF8) and verify co-detection with both tag-specific and SCAF8-specific antibodies

This validation approach is particularly important for SCAF8 given its sequence similarity to SCAF4 and its multiple functional domains that may be differentially accessible in various experimental contexts.

How should results from SCAF8 knockout experiments be interpreted in the context of RNA processing?

When interpreting SCAF8 knockout experiment results in RNA processing studies:

• Distinguish between redundant and unique functions:

  • SCAF8 single KO effects are likely masked by SCAF4 redundancy for anti-termination

  • Focus on SCAF8-specific phenotypes related to transcript elongation

  • The most dramatic effects on alternative polyadenylation will only appear in double SCAF4/8 KO models

• Key readouts to analyze:

  • Nascent RNA-seq data: Examine transcript elongation rates and efficiency

  • mRNA-seq data: Analyze for alternative last exon (ALE) usage

  • MISO analysis: Focus on alternative polyadenylation site selection rather than splicing changes

• Data interpretation framework:

  Experimental System	Expected Phenotype	Interpretation
  SCAF8 single KO	Minimal changes to polyA site selection	Redundancy with SCAF4 masks effects
  SCAF4 single KO	Increased transcriptional readthrough	SCAF8 promotes elongation in absence of SCAF4
  SCAF4/8 double KO	Dramatic increase in proximal polyA site usage	Essential anti-termination function revealed

• Protein-level consequences: Assess whether alternative polyA site usage results in truncated protein products lacking functional domains, which has been demonstrated in published studies for genes like ZC3HAV1 and USP15 .

The lethal phenotype of double KO cells is consistent with the accumulation of truncated, non-functional proteins across the proteome due to premature termination .

What are the implications of SCAF8-TIAM2 fusion detection in cancer samples for antibody-based studies?

The detection of SCAF8-TIAM2 fusion transcripts in ovarian and endometrial tumors has important implications for antibody-based studies:

• Epitope accessibility challenges:

  • C-terminal antibodies may fail to detect fusion proteins if that region is lost

  • N-terminal antibodies might detect both wild-type SCAF8 and fusion proteins

  • Solution: Use antibodies targeting different regions of SCAF8 to differentiate between full-length and fusion proteins

• Distinguishing genomic fusions from readthrough events:

  • Some apparent SCAF8-TIAM2 fusion transcripts occur without genomic evidence

  • These likely represent readthrough transcription events

  • TIAM2 induction is lower in readthrough cases compared to genomic deletion cases

  • Solution: Combine antibody detection with genomic analysis

• Tissue-specific considerations:

  • Wild-type SCAF8 has highest expression in ovary and uterus

  • This makes it an ideal fusion partner to drive high expression in gynecological cancers

  • Solution: Consider tissue context when interpreting antibody staining patterns

• Methodological recommendations:

  • Combine immunoblotting with RT-PCR for fusion transcript detection

  • Use antibodies targeting the N-terminal portion of SCAF8 that would be retained in fusion proteins

  • Include controls from multiple tissue types to establish baseline expression patterns

Understanding these fusion events provides insight into tissue-specific oncogenic mechanisms and highlights the need for comprehensive validation when using SCAF8 antibodies in cancer research.

How can SCAF8 antibodies be utilized in PAR-CLIP experiments to map RNA binding sites?

For mapping SCAF8 RNA binding sites with PAR-CLIP (Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation):

• Experimental design optimization:

  • Incorporate 4-thiouridine (4SU) into nascent RNA (typically 100-500 μM for 14-16 hours)

  • UV crosslink at 365 nm (optimal for 4SU)

  • Perform stringent immunoprecipitation with SCAF8 antibodies

  • Look for characteristic T→C transitions in sequencing data that indicate direct RNA-protein crosslinks

• Key controls and validation approaches:

  • Parallel IP with untagged cell lines or IgG control

  • Replicate analysis (identify clusters appearing in 2-3 biological replicates)

  • Motif discovery analysis to identify sequence preferences

  • Comparative analysis with SCAF4 PAR-CLIP data

• Analysis guidelines based on published findings:

  • Focus on clusters appearing within annotated transcripts (>95% of SCAF8 binding sites)

  • Look for enrichment near splice junctions (SCAF8 binding is significantly enriched near these sites)

  • Analyze distance to intronic polyadenylation (IpA) sites

  • Examine correlation with RNAPII occupancy data

Research has demonstrated that SCAF8 and SCAF4 bind remarkably similar RNA targets, with 65% of SCAF8 binding sites overlapping with SCAF4 binding sites, despite being functionally distinct in some contexts .

What methodological approaches can reveal the structural basis of SCAF8 interaction with phosphorylated RNA polymerase II CTD?

To investigate the structural basis of SCAF8 interaction with phosphorylated RNAPII CTD:

• In vitro binding assays with purified components:

  • Express and purify full-length SCAF8 or the isolated CID domain

  • Use chemically phosphorylated CTD peptides with different modification patterns

  • Employ peptide binding assays to quantify interaction strengths

  • Published research shows strong preference for Ser2-Ser5 double-phosphorylated CTD peptides

• Structural biology approaches:

  • X-ray crystallography of SCAF8 CID domain with phosphorylated CTD peptides

  • Cryo-EM of larger complexes

  • NMR for dynamic interaction studies

  • Comparative analysis with SCAF4 and other CID-containing proteins

• Mutational analysis:

  • Generate point mutations in the SCAF8 CID domain

  • Test effects on CTD binding using co-IP experiments

  • Examine functional consequences in cellular assays

• Phosphorylation-specific interaction analysis:

  • Compare binding to different CTD phosphorylation patterns:

    • Ser2P only

    • Ser5P only

    • Ser2P-Ser5P

    • Tyr1P-Ser2P

  • Research shows that Tyr1 phosphorylation reduces binding relative to Ser2 phosphorylation alone

These approaches can reveal how SCAF8 specifically recognizes the Ser2-Ser5 bi-phosphorylated CTD, which is critical for understanding its role in coordinating transcription elongation and termination.

This detailed methodological framework builds upon published research demonstrating that SCAF8 preferentially binds RNAPII with specific CTD phosphorylation patterns, informing both the mechanistic understanding of transcriptional regulation and the development of targeted experimental approaches.