SPCC825.05c Antibody

What is SPCC825.05c and what is its role in fission yeast?

SPCC825.05c is a protein in Schizosaccharomyces pombe (fission yeast) that has been identified as Saf5, a factor involved in splicing regulation. Recent systematic screening has revealed that Saf5 serves as an important link between splicing and transcription processes . The protein is part of the NineTeen Complex (NTC), which plays a crucial role in the splicing mechanism of pre-mRNAs. Studies indicate that SPCC825.05c/Saf5 particularly affects splicing when one intron of a given pre-mRNA is affected, suggesting that it coordinates transcription rates with splicing efficiency .

What experimental systems are most appropriate for studying SPCC825.05c function?

The most suitable experimental system for studying SPCC825.05c function is the fission yeast S. pombe model system, which offers several advantages:

• S. pombe contains introns in 43% of its genes, making it an excellent model for studying splicing mechanisms

• Genetic manipulation techniques are well-established in fission yeast

• Flow cytometry can be used to analyze cell cycle progression in S. pombe strains with SPCC825.05c mutations

• Genetically-encoded splicing reporters have been developed to quantify splicing efficiency in vivo in intact yeast cells

Researchers should consider implementing reporter systems containing fluorescent proteins (such as mRFP and YFP) flanking the gene of interest, as this allows for direct visualization of splicing events when studying SPCC825.05c .

How should SPCC825.05c antibody specificity be validated for research applications?

To validate the specificity of SPCC825.05c antibody, researchers should implement a multi-step approach:

• Knockout/deletion control testing: Use Δsaf5 strains alongside wild-type controls to verify absence of signal in knockout samples

• Western blot validation: The antibody should recognize a band at the expected molecular weight of SPCC825.05c. Implement the following controls:

  • Include positive controls using recombinant SPCC825.05c protein

  • Test antibody on wild-type and SPCC825.05c deletion strains

  • Use appropriate loading controls (like anti-Sty1)

• Cross-reactivity assessment: Test the antibody against related proteins within the splicing complex to ensure specificity

• Epitope analysis: When available, use different antibodies targeting distinct epitopes of SPCC825.05c to confirm results

• Biopanning approaches: Consider using yeast surface display methods for high-specificity validation as described in recent protocols for phosphorylation-specific antibodies

What controls are essential when designing experiments using the SPCC825.05c antibody?

When designing experiments with SPCC825.05c antibody, the following controls are critical:

• Positive controls:

  • Wild-type S. pombe strains expressing SPCC825.05c

  • Recombinant SPCC825.05c protein (when available)

  • Strains overexpressing tagged SPCC825.05c (sfGFP-tagged proteins can be used as reference)

• Negative controls:

  • SPCC825.05c deletion strains (Δsaf5)

  • Secondary antibody-only controls

  • Isotype controls

• Loading/normalization controls:

  • Anti-Sty1 polyclonal antibody can be used as a loading control

  • For immunofluorescence, DAPI nuclear stain should be included

• Technical controls:

  • Include multiple biological and technical replicates

  • Implement proper randomization and blinding procedures in quantitative analyses

How can SPCC825.05c antibody help investigate the link between splicing and transcription?

SPCC825.05c antibody can be instrumental in investigating the splicing-transcription link through several methodological approaches:

• Chromatin Immunoprecipitation (ChIP): Use the antibody to pull down SPCC825.05c and identify its associated genomic regions, revealing potential co-localization with transcription machinery

• Co-immunoprecipitation (Co-IP): Apply the antibody to identify protein interactions between SPCC825.05c and transcription factors or other splicing regulators

• Simultaneous tracking: Combine SPCC825.05c antibody with RNA polymerase II antibodies to track the co-localization during active transcription

• Splicing reporter assays: As described in recent studies, researchers can use fluorescent reporter constructs to quantify splicing efficiency in SPCC825.05c mutant backgrounds compared to wild-type :

• Intron retention analysis: RNA-seq data from SPCC825.05c-depleted cells reveals that when one intron in a transcript is affected, other introns in the same transcript are also likely to be affected, suggesting a global role in coordinating splicing with transcription rate

What are the methodological approaches for studying SPCC825.05c's role in the NineTeen Complex (NTC)?

Researchers investigating SPCC825.05c's role in the NTC should consider these methodological approaches:

• Comparative mutation analysis: Compare phenotypes between Δsaf5 and mutations in other NTC components (like Δcwf12) to establish functional relationships

• Domain-specific antibodies: Use antibodies targeting different regions of SPCC825.05c to determine which domains interact with other NTC components

• Quantitative proteomics:

  • Immunoprecipitate SPCC825.05c using validated antibodies

  • Analyze associated proteins by mass spectrometry

  • Compare NTC composition in wild-type versus stress conditions

• Function-specific assays: Implement splicing reporter constructs with different splicing site mutations (5'SS, BP) to determine how SPCC825.05c influences recognition of specific splicing signals

• Temporal dynamics analysis: Track NTC assembly and disassembly using pulse-chase experiments with SPCC825.05c antibody

What are the optimal protocols for Western blot detection of SPCC825.05c?

For optimal Western blot detection of SPCC825.05c, researchers should follow these methodological considerations:

• Sample preparation:

  • Extract proteins using TCA precipitation, as described in published protocols

  • Use fresh samples whenever possible to avoid protein degradation

• Gel selection:

  • Use 4-20% Tris-Glycine gradient gels for optimal resolution

  • Single percentage gels should be selected based on SPCC825.05c's molecular weight according to this table :

  Gel Type	Protein Molecular Weight
  3-8% Tris-Acetate	> 200 kDa
  4-20% Tris-Glycine	10-200 kDa
  10-20% Tris-Glycine	< 100 kDa

• Antibody conditions:

  • Primary antibody dilution: Optimize starting at 1:1000 for polyclonal antibodies (similar to p53 antibody protocols)

  • Incubation: 2 hours at room temperature or overnight at 4°C

  • Secondary antibody: Use goat anti-rabbit IgG:HRP at 1:5000

• Detection optimization:

  • For chemiluminescent detection: Expose for 10 seconds initially, then adjust as needed

  • Consider TMB solution development for 12 minutes at room temperature as an alternative method

• Controls:

  • Always include wild-type and Δsaf5 controls on the same gel

  • Use anti-Sty1 polyclonal antibody as loading control on the same membrane

How can flow cytometry be optimized for studies involving SPCC825.05c antibody?

When optimizing flow cytometry for SPCC825.05c-related studies, implement these methodological approaches:

• Sample preparation:

  • Culture strains in 96-well plates using a 96-pin replicator for consistency

  • Include JA2922, JA2920, and wild-type strains as controls in specific wells (e.g., H2, H3, H12)

• Instrument setup:

  • Use BD LSRFortessa™ flow cytometer with High Throughput Sampler (HTS)

  • For fluorescent reporter detection, configure for both YFP and mRFP channels

• Gating strategy:

  • Implement the pulse-width versus forward scatter methodology to exclude cell doublets, which is especially important for fission yeast cells that tend to form multimers

  • Gate on mononuclear cells, as described in published protocols for cell-cycle analysis

• Data analysis:

  • Calculate splicing efficiency using the ratio between YFP and mRFP signals in splicing reporter systems

  • Apply appropriate compensation to account for spectral overlap

• Quantification:

  • Compare splicing efficiencies between wild-type, Δsaf5, and other splicing factor mutations

  • Statistical analysis should include at least three biological replicates

How can SPCC825.05c antibody studies be integrated with RNA-seq and transcriptome analysis?

To integrate SPCC825.05c antibody studies with RNA-seq and transcriptome analysis, researchers should:

• Immunoprecipitation followed by RNA-seq (RIP-seq):

  • Immunoprecipitate SPCC825.05c-RNA complexes using validated antibodies

  • Sequence associated RNAs to identify direct targets

  • Compare wild-type vs. Δsaf5 transcriptomes to identify affected splicing events

• Single-cell approaches:

  • Combine SPCC825.05c antibody staining with single-cell RNA sequencing

  • Consider adaptation of FB5P-seq (FACS-based 5'-end single-cell RNA sequencing) protocols that allow for both transcriptome analysis and antibody detection

• Analysis of intron retention:

  • Focus RNA-seq analysis on intron retention events, particularly examining whether multiple introns in the same transcript are affected in Δsaf5 mutants

  • Calculate Percent Intron Retention (PIR) values for each intron

• Public antibody sequence database integration:

  • Utilize databases of antibody sequences like those described in search result to identify public antibody responses

  • Compare binding patterns with known splicing factors

• Deep learning approaches:

  • Apply deep learning models similar to those used in antibody research to predict SPCC825.05c binding sites and functional impacts

What methodological approaches can be used to study SPCC825.05c's role during the cell cycle?

To study SPCC825.05c's role during the cell cycle, researchers should implement these methodological approaches:

• Synchronized cell cultures:

  • Use established protocols to synchronize fission yeast cultures

  • Take time points at different cell cycle stages for SPCC825.05c antibody analysis

• Cell cycle-specific analysis:

  • Combine SPCC825.05c antibody detection with DNA content analysis to distinguish G1, S, and G2/M phases

  • Apply the flow cytometry method for cell-cycle analysis in fission yeast described in search result , which allows discrimination between G1 and G2 phases

• Integration with cell cycle-regulated gene expression data:

  • Compare SPCC825.05c binding patterns with known cell cycle-regulated genes in fission yeast

  • Analyze whether splicing patterns of cell cycle-regulated genes are differentially affected by SPCC825.05c deletion

• Microscopy approaches:

  • Use immunofluorescence with SPCC825.05c antibody to track protein localization during cell cycle

  • Implement this protocol for optimal results:

    • Fixation: 4% Formaldehyde for 15 min at RT

    • Primary antibody: Optimized dilution (start at 1:100)

    • Secondary antibody: Goat Anti-Rabbit ATTO 488 at 1:100

    • Counterstain: DAPI nuclear stain at 1:5000

• Cell cycle-specific splicing reporter assays:

  • Deploy the fluorescent splicing reporters at different cell cycle stages

  • Quantify how SPCC825.05c affects splicing efficiency in a cell cycle-dependent manner

How can non-specific binding issues with SPCC825.05c antibody be addressed?

To address non-specific binding issues with SPCC825.05c antibody, implement these methodological solutions:

• Buffer optimization:

  • Use storage buffer containing 50% Glycerol, 0.01M PBS, pH 7.4 with 0.03% Proclin 300 as preservative

  • For blocking, use 5% skim milk in TBST for optimal results

• Dilution series testing:

  • Perform titration experiments to determine optimal antibody concentration

  • Start with 1:500, 1:1000, and 1:2000 dilutions to find the best signal-to-noise ratio

• Exclusion of aggregates:

  • When analyzing antigen or antibody aggregates, implement the aggregate exclusion methodology described in Cell Ranger outputs

  • Use the UMI correction metrics to identify potential aggregate formation

• Cross-adsorption:

  • Pre-adsorb antibody with cell lysates from Δsaf5 strains to remove non-specific antibodies

  • Use protein A beads for immunoprecipitation and mass spectrometry to confirm specific targeting

• Validation with competitive binding assay:

  • Incubate the antibody with purified SPCC825.05c protein before application to verify that signal disappears in competitive binding conditions

What are the most common technical challenges when working with SPCC825.05c antibody and how can they be overcome?

Common technical challenges and their methodological solutions include:

• Low signal strength:

  • Increase antibody concentration gradually

  • Extend primary antibody incubation time to overnight at 4°C

  • Use signal amplification methods such as biotin-streptavidin systems

  • Consider using more sensitive chemiluminescent substrates

• Inconsistent results between experiments:

  • Implement rigid standardization protocols

  • Prepare master mixes of antibody dilutions

  • Use automated pipetting systems when available

  • Store antibody in single-use aliquots at -80°C to avoid freeze-thaw cycles

• Difficulty detecting post-translational modifications:

  • Follow specific treatments that activate particular post-translational modifications

  • Consult resources like PhosphoSitePlus® for modifications of SPCC825.05c

  • Use modification-specific antibodies when available

• Batch effects in high-throughput experiments:

  • Include standardized controls in each batch

  • Implement plate normalization techniques

  • Consider using methods like 96-well plate whole-well image analysis for validation

• Signal detection in specific subcellular compartments:

  • For immunofluorescence, use specific subcellular markers alongside SPCC825.05c antibody

  • Expected localization: Cytoplasm, PML body, Endoplasmic Reticulum

  • Optimize fixation protocols for preserving nuclear structures

How might SPCC825.05c antibody be used to explore evolutionary conservation of splicing mechanisms?

To explore evolutionary conservation of splicing mechanisms using SPCC825.05c antibody:

• Comparative analysis across species:

  • Test cross-reactivity of SPCC825.05c antibody with homologs in other yeast species

  • Compare functional consequences of splicing factor mutations across evolutionary diverse organisms

• Domain-specific conservation studies:

  • Use different SPCC825.05c antibodies targeting specific domains to determine which regions are functionally conserved

  • Map these to known human splicing factor domains

• Public antibody response analysis:

  • Apply methodologies similar to those used for studying public antibody responses to SARS-CoV-2

  • Analyze recurring molecular features in antibodies targeting conserved splicing factors

• Design of conserved epitope antibodies:

  • Apply deep learning approaches like those in DyAb to design antibodies targeting evolutionarily conserved epitopes

  • Test these antibodies across multiple species to validate splicing mechanism conservation

• Multi-omics integration:

  • Combine antibody-based detection with genomic, transcriptomic and proteomic data to build comprehensive models of splicing conservation

  • Focus particularly on NTC components across species

What novel applications of SPCC825.05c antibody might emerge in disease-related research?

Potential novel applications in disease-related research include:

• Cancer splicing regulation models:

  • Use knowledge gained from SPCC825.05c studies to investigate human homologs in cancer cells

  • Apply antibodies against human counterparts to study splicing dysregulation in tumors

  • Draw parallels to p53 antibody applications in cancer research

• Autoimmune disease connections:

  • Explore whether autoantibodies against splicing factors play a role in diseases like rheumatoid arthritis

  • Apply methodologies similar to those used in identifying anticitrullinated SR-A peptide antibodies

• Neurological disorder implications:

  • Investigate whether mutations in human homologs of SPCC825.05c contribute to neurological disorders with known splicing defects

  • Develop antibodies specific to disease-associated variants

• Therapeutic antibody development:

  • Apply high-throughput screening approaches like those described for antibody development

  • Design therapeutic antibodies targeting disease-specific splicing factor conformations

  • Use yeast biopanning approaches to develop highly specific antibodies

• Diagnostic applications:

  • Develop antibody-based diagnostic tests for diseases with splicing factor abnormalities

  • Apply machine learning to predict antibody characteristics and specificity