SPAC9E9.05 Antibody

What is SPAC9E9.05 and what is its functional significance?

SPAC9E9.05 is a gene in Schizosaccharomyces pombe (fission yeast) that encodes a Sororin-like protein now designated as Sor1. Initially annotated as a poorly characterized Schizosaccharomyces-specific protein, it has been identified as an ortholog of metazoan Sororin, involved in sister chromatid cohesion regulation. The protein contains a conserved Sororin domain with two critical phenylalanine residues and a K/R-rich domain at its C-terminus that are essential for cohesion maintenance . Deletion mutants of SPAC9E9.05 exhibit negative synthetic growth interactions with cohesion-defective mutants (eso1-G799D and mis4-242), supporting its role in chromosome cohesion regulation .

How does Sor1 (SPAC9E9.05) compare structurally to mammalian Sororin?

While SPAC9E9.05 (Sor1) shows limited sequence homology with vertebrate Sororin, both share distinguishing structural features:

What are the recommended approaches for generating specific antibodies against SPAC9E9.05/Sor1?

When generating antibodies against SPAC9E9.05, researchers should consider the following methodological approach:

• Epitope selection: The C-terminal Sororin domain containing the two conserved phenylalanine residues offers a distinctive region for antibody generation. Alternatively, the K/R-rich domain presents another unique target .

• Expression and purification strategy: Express the full-length protein or specific domains as recombinant fusion proteins (common tags include GST, MBP, or His-tags) in bacterial systems. For challenging expression, consider using a ubiquitin fusion system similar to that described for other S. pombe proteins .

• Immunization protocol: Implement a standard immunization schedule in rabbits (for polyclonal) or mice (for monoclonal) with appropriate adjuvant selection. Polyacrylate-based adjuvants have shown efficacy for enhancing antibody responses to recombinant proteins .

• Antibody purification: Purify using affinity chromatography with immobilized antigen, followed by cross-adsorption against lysates from SPAC9E9.05 deletion strains to remove non-specific antibodies .

How should researchers validate a newly generated SPAC9E9.05/Sor1 antibody?

A comprehensive validation strategy should include:

• Western blot analysis: Compare signal between wild-type and SPAC9E9.05Δ strains. Expected molecular weight would be approximately 30 kDa based on protein size .

• Immunoprecipitation followed by mass spectrometry: Confirm that the antibody pulls down SPAC9E9.05 and its known interactors (Pds5, cohesin components) .

• Immunofluorescence microscopy: Verify subcellular localization patterns and absence of signal in deletion strains.

• Co-immunoprecipitation assays: Validate by demonstrating interaction with known binding partners like Pds5-Myc and Psm3-GFP (S. pombe cohesin subunit), as demonstrated for Sor1-Pk in previous studies .

• Epitope competition assay: Preincubate antibody with excess purified antigen or synthetic peptide representing the epitope to confirm specificity .

What is the optimal protocol for detecting SPAC9E9.05/Sor1 by immunoblotting?

For effective immunoblot detection of SPAC9E9.05:

• Sample preparation:

  • Harvest 1-5 × 10^7 S. pombe cells

  • Lyse cells using glass bead disruption in buffer containing protease inhibitors

  • Clarify lysate by centrifugation (14,000 × g, 10 min, 4°C)

• Protein separation:

  • Load 20-50 μg of total protein per lane

  • Use 12% SDS-PAGE gels for optimal resolution of SPAC9E9.05 (~30 kDa)

• Transfer conditions:

  • Transfer to PVDF membrane (100V for 1 hour or 30V overnight)

  • Verify transfer efficiency with Ponceau S staining

• Immunodetection:

  • Block with 5% non-fat milk or BSA in TBST for 1 hour

  • Incubate with primary antibody (initial dilution range: 1:500-1:2000)

  • Wash 3× with TBST

  • Incubate with HRP-conjugated secondary antibody (e.g., goat anti-mouse IgG HRP at 1:5000)

  • Develop using enhanced chemiluminescence

• Controls:

  • Include SPAC9E9.05 deletion strain as negative control

  • Consider including epitope-tagged version (e.g., Sor1-Pk) as positive control

How can SPAC9E9.05/Sor1 antibodies be effectively used in immunoprecipitation studies?

For coimmunoprecipitation (co-IP) experiments to study Sor1 interactions:

• Cell lysis conditions:

  • Harvest 5 × 10^8 cells from mid-log phase culture

  • Lyse in buffer containing: 50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, 10% glycerol, supplemented with protease and phosphatase inhibitors

• Pre-clearing:

  • Incubate lysate with Protein A/G beads for 1 hour at 4°C

  • Remove beads by centrifugation

• Immunoprecipitation:

  • Add SPAC9E9.05 antibody (2-5 μg) to pre-cleared lysate

  • Incubate overnight at 4°C with gentle rotation

  • Add Protein A/G beads for 2 hours

  • Wash 4× with lysis buffer

• Elution and analysis:

  • Elute proteins with SDS sample buffer at 95°C for 5 minutes

  • Analyze by Western blotting for Sor1 and potential interactors like Pds5-Myc and Psm3-GFP

• Controls:

  • IgG control antibody for non-specific binding

  • Input sample (5-10% of lysate)

  • SPAC9E9.05 deletion strain

What are the recommended procedures for using SPAC9E9.05/Sor1 antibodies in immunofluorescence microscopy?

For optimal immunofluorescence results with S. pombe cells:

• Cell fixation:

  • Grow cells to mid-log phase in appropriate medium

  • Fix with 3.7% formaldehyde for 30 minutes or cold methanol for 10 minutes

  • For membrane proteins, consider mixed aldehyde fixation (formaldehyde + glutaraldehyde)

• Cell wall digestion:

  • Treat with zymolyase (0.5 mg/ml) or lysing enzymes in sorbitol buffer for 10-30 minutes

  • Monitor cell wall digestion microscopically

• Permeabilization:

  • Permeabilize with 0.1% Triton X-100 for 5 minutes

• Blocking and antibody incubation:

  • Block with 1% BSA in PBS for 30 minutes

  • Incubate with primary antibody (start with 1:100 dilution) overnight at 4°C

  • Wash 3× with PBS + 0.1% Tween-20

  • Incubate with fluorescent secondary antibody (1:500) for 1 hour at room temperature

  • Counterstain DNA with DAPI (1 μg/ml)

• Imaging considerations:

  • Based on Sor1's role in sister chromatid cohesion, focus on nuclear localization during different cell cycle stages

  • Co-stain with cohesin subunits to observe colocalization

How can SPAC9E9.05/Sor1 antibodies be utilized in chromatin immunoprecipitation (ChIP) experiments?

For studying Sor1 chromatin association:

• Crosslinking and chromatin preparation:

  • Crosslink 5 × 10^8 cells with 1% formaldehyde for 15 minutes at room temperature

  • Quench with 125 mM glycine for 5 minutes

  • Wash cells and lyse in buffer containing protease inhibitors

  • Sonicate to generate chromatin fragments of 200-500 bp

• Immunoprecipitation:

  • Pre-clear chromatin with Protein A/G beads

  • Incubate with SPAC9E9.05 antibody (5 μg) overnight at 4°C

  • Collect complexes with Protein A/G beads

  • Wash sequentially with low salt, high salt, LiCl, and TE buffers

• Reversal of crosslinking and DNA purification:

  • Reverse crosslinks at 65°C for 6 hours

  • Treat with RNase A and Proteinase K

  • Purify DNA using phenol-chloroform extraction or commercial kits

• Analysis options:

  • qPCR for specific genomic regions (such as centromeres, cohesin-associated regions)

  • ChIP-seq for genome-wide binding profile

• Controls and validation:

  • Input chromatin (10%)

  • IgG control antibodies

  • Positive control (antibody against known cohesin component)

  • ChIP in SPAC9E9.05 deletion strain

What strategies can be employed to investigate Sor1 post-translational modifications using antibodies?

To examine potential post-translational modifications (PTMs) of Sor1:

• Phosphorylation analysis:

  • Immunoprecipitate Sor1 from cells treated with or without phosphatase inhibitors

  • Run Phos-tag SDS-PAGE to detect mobility shifts

  • Perform Western blotting with phospho-specific antibodies if available

  • Consider mass spectrometry analysis of immunoprecipitated Sor1 for phosphosite mapping

• Ubiquitination analysis:

  • Express Ub-GFP-Sor1 fusion constructs using vectors similar to pREP41-Ub-GFP-SpCPS

  • Immunoprecipitate under denaturing conditions to preserve ubiquitination

  • Probe with anti-ubiquitin antibodies

  • Compare ubiquitination patterns in wild-type vs. mutant backgrounds

• Cell cycle-dependent modification:

  • Synchronize cells using standard methods for S. pombe

  • Collect samples at different cell cycle stages

  • Analyze by immunoblotting for mobility shifts

  • Perform phosphatase treatment to confirm phosphorylation

• Mass spectrometry approach:

  • Large-scale immunoprecipitation of Sor1

  • Tryptic digestion and LC-MS/MS analysis

  • Identify PTM sites by peptide mapping

How can researchers investigate the dynamics of SPAC9E9.05/Sor1 recruitment during DNA damage response?

To study Sor1's potential role in DNA damage response:

• DNA damage induction:

  • Treat cells with various damaging agents (UV, MMS, hydroxyurea, ionizing radiation)

  • Include time course experiments (15 min, 30 min, 1 hour, 2 hours post-damage)

• Chromatin fractionation:

  • Separate soluble and chromatin-bound fractions

  • Analyze Sor1 distribution by immunoblotting

  • Compare with known DNA damage response proteins

• Immunofluorescence microscopy:

  • Visualize Sor1 localization before and after DNA damage

  • Co-stain with γH2AX or Rad51 to mark damage sites

  • Quantify colocalization with damage markers

• ChIP-seq analysis:

  • Perform ChIP-seq in untreated and damaged cells

  • Analyze changes in Sor1 binding patterns

  • Compare with binding profiles of cohesin and DNA repair factors

• Protein interactions during damage:

  • Conduct co-IP experiments before and after damage induction

  • Identify damage-specific interaction partners by mass spectrometry

  • Validate key interactions by reciprocal co-IP

What approaches can be used to study the functional relationship between Sor1 and WAPL using antibodies?

To investigate whether Sor1, like vertebrate Sororin, antagonizes WAPL activity:

• Co-immunoprecipitation studies:

  • Generate antibodies against S. pombe WAPL homolog (Wpl1)

  • Perform reciprocal co-IPs to detect physical interactions

  • Compare complex formation in different cell cycle stages

• Proximity ligation assay (PLA):

  • Use antibodies against Sor1 and Wpl1 for in situ PLA

  • Quantify interaction signals throughout the cell cycle

  • Compare with cohesin subunit interactions

• Competitive binding assays:

  • Immunoprecipitate Pds5 and probe for Sor1 and Wpl1

  • Test if overexpression of one displaces the other

  • Use recombinant proteins for in vitro competition assays

• Genetic interaction studies:

  • Create strains with tagged versions of both proteins

  • Analyze phenotypes of single and double mutants

  • Use antibodies to examine protein levels and localization in these backgrounds

• Domain-specific antibodies:

  • Generate antibodies against specific domains (e.g., Sororin domain, FGF motif)

  • Use these to map interaction interfaces

  • Perform peptide competition assays to confirm domain specificity