SPCC417.09c Antibody

What is SPCC417.09c and what functional roles does it play in experimental systems?

SPCC417.09c is an uncharacterized transcriptional regulatory protein found in the fission yeast Schizosaccharomyces pombe, with a full length of 767 amino acids . While its complete functional characterization remains ongoing, it is classified as a potential transcriptional regulatory protein based on sequence analysis.

The protein's regulatory functions can be studied through various immunological approaches. When designing experiments involving SPCC417.09c, researchers should consider:

• Protein localization studies to determine subcellular distribution

• Interaction partners identification through co-immunoprecipitation

• Transcriptional activity assessment through reporter assays

• DNA-binding capacity evaluation via ChIP-seq approaches

Current experimental evidence suggests SPCC417.09c may function analogously to other transcriptional regulatory proteins in yeast systems, with potential roles in stress response pathways or developmental programs.

How can researchers validate the specificity of anti-SPCC417.09c antibodies?

Validating antibody specificity is essential for generating reliable experimental data. For SPCC417.09c antibodies, implement the following comprehensive validation protocol:

• Genetic validation approach:

  • Compare wild-type S. pombe expressing SPCC417.09c with SPCC417.09c deletion strains

  • Observe presence of signal in wild-type and absence in knockout strains

• Biochemical validation methods:

  • Perform Western blot analysis to confirm detection at the expected molecular weight (~84 kDa)

  • Conduct pre-absorption tests by pre-incubating antibody with purified recombinant SPCC417.09c protein

  • Implement peptide competition assays to verify binding specificity

• Advanced validation techniques:

  • Employ immunoprecipitation followed by mass spectrometry

  • Evaluate cross-reactivity against related yeast proteins

  • Test antibody performance across multiple experimental conditions

Similar validation approaches have been successfully employed with other antibodies, such as those against polyomaviruses, where cross-reactivity testing is essential for experimental reliability .

What are the optimal conditions for Western blot analysis using anti-SPCC417.09c antibody?

For robust and reproducible Western blot detection of SPCC417.09c protein:

• Sample preparation:

  • Implement glass bead lysis in buffer containing 50mM Tris-HCl pH 7.5, 150mM NaCl, 0.5% NP-40, 1mM EDTA

  • Include complete protease inhibitor cocktail to prevent degradation

  • Load 20-50μg total protein per lane for adequate signal detection

• Electrophoresis parameters:

  • Use 8-10% SDS-PAGE gels for optimal resolution of SPCC417.09c (~84 kDa)

  • Run gel at 100V through stacking gel, then 150V through resolving gel

  • Include molecular weight markers spanning 50-100 kDa range

• Transfer optimization:

  • For proteins >70 kDa, employ wet transfer at 30V overnight at 4°C

  • Use 0.45μm PVDF membrane rather than 0.2μm for larger proteins

  • Verify transfer efficiency with reversible protein stain before blocking

• Antibody incubation conditions:

  • Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

  • Test primary antibody dilutions from 1:500 to 1:2000

  • Incubate primary antibody overnight at 4°C for maximum sensitivity

  • Use HRP-conjugated secondary antibodies at 1:5000 dilution

This approach aligns with methodologies used for other difficult-to-detect transcriptional regulators and has shown consistent results across independent laboratory validations .

How can researchers distinguish between false negative results and true absence of SPCC417.09c expression?

When working with potentially low-abundance transcriptional regulators like SPCC417.09c, distinguishing between technical failures and true biological absence requires systematic methodology:

• Technical verification steps:

  • Include positive controls (recombinant SPCC417.09c protein) on every blot

  • Verify protein extraction efficiency through detection of abundant housekeeping proteins

  • Implement multiple detection methods (chemiluminescence, fluorescence) with varying sensitivity

• Biological verification approaches:

  • Perform RT-PCR to confirm SPCC417.09c mRNA expression

  • Generate epitope-tagged SPCC417.09c constructs as alternative detection method

  • Evaluate expression under conditions known to upregulate transcriptional regulators (stress, cell cycle arrest)

• Antibody performance assessment:

  • Test multiple antibody preparations targeting different epitopes

  • Employ signal amplification systems for low-abundance proteins

  • Compare monoclonal versus polyclonal antibody performance

Systematic troubleshooting reveals that apparent absence of detection can frequently be attributed to technical factors rather than true absence of the target protein. When antibodies fail to detect their targets despite confirmed gene expression, consider epitope masking or protein conformational issues that may interfere with antibody binding .

What methodological approaches can optimize immunoprecipitation studies with anti-SPCC417.09c antibodies?

For successful immunoprecipitation of SPCC417.09c and associated complexes:

• Lysis buffer optimization:

  • For nuclear proteins like transcriptional regulators, include 0.1% SDS or 0.1% sodium deoxycholate

  • Test multiple detergent conditions (NP-40, Triton X-100, CHAPS) at 0.1-1% concentration

  • For DNA-binding proteins, include DNase I treatment to release chromatin-bound factors

• Technical parameters for maximum recovery:

  • Use 2-5μg antibody per 500μg total protein

  • Pre-clear lysates with Protein A/G beads for 1 hour at 4°C

  • Extend antibody binding to overnight at 4°C with gentle rotation

  • Implement stringent washing (4× lysis buffer, 1× PBS) to reduce background

• Validation and controls:

  • Include IgG control from same species as the primary antibody

  • Save 5-10% of pre-IP lysate as input control

  • Perform parallel IPs with epitope-tagged version of SPCC417.09c

• Analysis of co-immunoprecipitated complexes:

  • Silver stain for visualization of interacting proteins

  • Mass spectrometry for unbiased identification of protein complexes

  • Western blot for validation of specific protein-protein interactions

This methodology has been successfully applied to study protein complexes involved in transcriptional regulation and can be adapted specifically for SPCC417.09c investigations .

How do post-translational modifications affect detection of SPCC417.09c by antibodies?

Post-translational modifications (PTMs) can significantly impact antibody recognition of transcriptional regulators like SPCC417.09c:

• Common PTMs affecting antibody recognition:

  • Phosphorylation can alter epitope accessibility or create charge-based interference

  • Ubiquitination may mask epitopes or create steric hindrance

  • Proteolytic processing can remove epitopes entirely from processed forms

• Experimental strategies to address PTM interference:

  • Generate antibodies against multiple regions of SPCC417.09c

  • Use phosphatase treatment to remove phosphorylation

  • Implement deubiquitinating enzyme treatment prior to analysis

  • Compare native versus denaturing conditions for epitope accessibility

• Detection of modified forms:

  • Look for mobility shifts on Western blots indicating PTMs

  • Use Phos-tag™ gels to separate phosphorylated from non-phosphorylated forms

  • Employ 2D gel electrophoresis to resolve differently modified protein species

Lessons from antibody studies in other systems demonstrate that recognition of target proteins can be dramatically affected by their modification state. For instance, certain monoclonal antibodies against proteins like PD-L1 show variable staining intensity depending on the epitope's accessibility and modification state .

What are the optimal conditions for immunofluorescence studies of SPCC417.09c in yeast cells?

For high-quality immunofluorescence detection of SPCC417.09c in S. pombe:

• Fixation protocol optimization:

  • Test both formaldehyde (3.7%, 30 minutes) and methanol (-20°C, 6 minutes) fixation

  • For transcription factors, methanol often provides better nuclear epitope accessibility

  • After formaldehyde fixation, quench with 50mM NH₄Cl for 10 minutes

• Permeabilization conditions:

  • For formaldehyde-fixed cells: 1.2M sorbitol with 0.1% Triton X-100

  • For methanol-fixed cells: Additional permeabilization often unnecessary

  • For difficult epitopes: Test enzymatic digestion of cell wall with zymolyase

• Blocking and antibody parameters:

  • Block with 5% BSA or 5% normal serum from secondary antibody species

  • Test primary antibody dilutions from 1:50 to 1:500

  • Include DAPI or Hoechst staining to visualize nuclei

• Imaging considerations:

  • Use confocal microscopy for optimal subcellular localization

  • Implement Z-stack acquisition to capture complete cell volume

  • Consider deconvolution for improved signal-to-noise ratio

• Controls and validation:

  • Include peptide competition assay to verify staining specificity

  • Compare staining pattern with epitope-tagged SPCC417.09c

  • Include SPCC417.09c deletion strain as negative control

Similar approaches have been successfully applied for detecting low-abundance transcription factors in yeast and can be specifically optimized for SPCC417.09c localization studies .

How can researchers optimize ChIP-seq experiments to study SPCC417.09c binding to chromatin?

For successful ChIP-seq analysis of SPCC417.09c DNA-binding properties:

• Crosslinking optimization:

  • For transcription factors, test formaldehyde concentrations between 0.75-1.5%

  • Optimize crosslinking time (10-20 minutes) to maximize capture without over-crosslinking

  • For weak or transient interactions, consider dual crosslinking with DSG followed by formaldehyde

• Chromatin preparation parameters:

  • Sonication conditions: Optimize to achieve fragments of 200-500 bp

  • Monitor fragmentation by agarose gel electrophoresis

  • Target DNA concentration of 10-20 ng/μl after sonication

• Immunoprecipitation protocol:

  • Test antibody amounts between 2-10 μg per reaction

  • Include essential controls: IgG negative control, histone H3 positive control

  • For potentially low-abundance factors like SPCC417.09c, increase starting material (10⁸ cells)

• Library preparation considerations:

  • For transcription factors, implement PCR-free library preparation if possible

  • Minimize PCR cycles (8-12) to reduce amplification bias

  • Include spike-in controls for quantitative analysis

• Bioinformatic analysis pipeline:

  • Use MACS2 with appropriate p-value thresholds for peak calling

  • Implement MEME and HOMER for de novo motif discovery

  • Integrate with RNA-seq data to identify potential regulatory targets

This methodological framework has been successfully applied to transcription factor ChIP-seq studies and can be adapted specifically for SPCC417.09c binding site analysis .

How do different antibody clones against SPCC417.09c compare in terms of performance and specificity?

When evaluating multiple antibody clones against SPCC417.09c, systematic performance comparison is essential:

• Comparative analysis methodology:

  • Test all antibodies simultaneously under identical conditions

  • Evaluate across multiple applications (Western blot, IP, IF, ChIP)

  • Quantify specificity using signal-to-noise ratio measurements

• Performance metrics to consider:

  • Sensitivity: Minimum detectable amount of target protein

  • Specificity: Presence/absence of non-specific bands or staining

  • Reproducibility: Consistency across multiple experiments

  • Application versatility: Performance across different techniques

• Clone-specific characteristics:

  • Epitope locations affect performance in different applications

  • Monoclonal antibodies offer consistency but may be sensitive to epitope modifications

  • Polyclonal antibodies provide robustness but may have batch-to-batch variability

Studies comparing antibody performance in other systems have demonstrated significant variability between clones. For example, analysis of SP142 and 22C3 monoclonal antibodies showed markedly different staining patterns and sensitivity levels despite targeting the same protein .

What strategies can resolve conflicting results between different detection methods for SPCC417.09c?

When faced with contradictory results across different experimental techniques:

• Systematic method validation approach:

  • Validate each technique independently with appropriate controls

  • Identify technique-specific limitations that might affect SPCC417.09c detection

  • Implement orthogonal methods to corroborate findings

• Technical factors assessment:

  • Compare native versus denatured detection methods

  • Evaluate epitope accessibility differences between techniques

  • Consider sensitivity thresholds of each method (Western blot vs. mass spectrometry)

• Biological context evaluation:

  • Test for cell cycle-dependent expression or localization

  • Compare results under different growth conditions or stresses

  • Assess if post-translational modifications affect detection differently across methods

• Resolution framework:

  • Employ multiple antibodies targeting different epitopes

  • Generate epitope-tagged versions for alternative detection

  • Implement functional assays to confirm biological activity