SPAC186.06 Antibody

What is SPAC186.06 and why is it significant for fission yeast research?

SPAC186.06 is a gene located in the subtelomeric region of Schizosaccharomyces pombe (fission yeast). Based on genomic studies, SPAC186.06 is part of a cluster of genes (including SPAC186.04c, SPAC186.05c, and SPAC186.07c) that show interesting expression patterns in response to various cellular conditions . The significance of SPAC186.06 lies in its potential role in heterochromatin regulation, as it falls within regions that are bound by the heterochromatin protein Swi6 . The protein encoded by this gene has been studied in the context of subtelomeric gene regulation and epigenetic control mechanisms in S. pombe.

What applications is the SPAC186.06 Antibody validated for?

The SPAC186.06 Antibody (product code CSB-PA889244XA01SXV) has been validated for the following applications:

• ELISA (Enzyme-Linked Immunosorbent Assay)

• Western Blotting (WB)

Similar to other S. pombe antibodies in this product line, it's designed specifically for research applications involving the detection and quantification of SPAC186.06 protein in fission yeast samples . The antibody is produced through antigen affinity purification methods, ensuring specificity for its target protein.

What is the recommended storage protocol for SPAC186.06 Antibody?

For optimal performance and longevity, the SPAC186.06 Antibody should be stored according to these guidelines:

• Store at -20°C or -80°C upon receipt

• Avoid repeated freeze-thaw cycles which can denature antibody proteins

• The antibody is typically provided in a storage buffer containing 50% Glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative

• When not in use, keep aliquoted samples at recommended temperatures and protect from light

• For short-term use (1-2 weeks), storage at 4°C is acceptable but not optimal for long-term preservation

How should I optimize Western blot protocols for SPAC186.06 Antibody?

When optimizing Western blot protocols for SPAC186.06 Antibody, consider the following methodological approach:

Sample Preparation:

• Extract proteins from S. pombe using established protocols for yeast cell lysis

• Use a lysis buffer containing: PBS pH 7.0, 200mM NaCl, 0.5mM EGTA, 0.5mM EDTA, 0.1% Triton X100, protease inhibitor cocktail, and 1mM phenylmethylsulfonyl fluoride

• Break cells by bead beating with glass beads for approximately 45 minutes at 4°C

• Centrifuge for five minutes at 1000g and collect the supernatant

Electrophoresis and Transfer:

• Resolve approximately 20μg of total protein extract on SDS-PAGE using 10-15% acrylamide gels

• Transfer proteins to nitrocellulose membranes

• Block with 5% milk in TBST

Detection:

• Recommended dilution: 1:500-1:5000 (start with 1:1000 and adjust as needed)

• Incubate with primary antibody overnight at 4°C

• Wash thoroughly with TBST

• Incubate with appropriate secondary antibody conjugated to HRP

• Detect using ECL SuperSignal detection system

Optimization Tips:

• Run a dilution series of your antibody to determine optimal concentration

• Include positive and negative controls

• Consider using recombinant SPAC186.06 protein as a positive control

What are the considerations for using SPAC186.06 Antibody in Chromatin Immunoprecipitation (ChIP)?

For successful ChIP experiments using SPAC186.06 Antibody:

Sample Preparation:

• Grow S. pombe to OD600 ≈ 1 in appropriate medium

• Cross-link with 1.5ml formaldehyde (37% solution) for 15 minutes

• Quench formaldehyde with 2.5ml of 2.5M glycine for five minutes

• Harvest cells and wash with cold PBS

• Lyse cells in buffer containing: 50mM HEPES-KOH pH7.5, 140mM NaCl, 1mM EDTA, 1% Triton X100, 0.1% Na-Deoxycholic acid

Sonication and Immunoprecipitation:

• Sonicate six times for 10 seconds at 80% amplitude with one minute on ice between each cycle

• Centrifuge sonicated material for 30 minutes at 2500 rpm

• Pre-clear supernatant with protein A/G sepharose beads

• Add 2-5μg of SPAC186.06 Antibody and incubate overnight at 4°C

• Wash beads sequentially with:

  • Lysis buffer

  • Lysis buffer with 360 mM NaCl

  • Washing buffer (10mM Tris/HCl pH8, 0.25M LiCl, 0.5% NP40, 0.5% Na-Deoxycholic acid, 1mM EDTA)

  • TE buffer

DNA Recovery and Analysis:

• Elute DNA-protein complexes with elution buffer (50mM Tris/HCl pH8, 10mM EDTA, 1% SDS) overnight at 65°C

• Purify DNA and analyze by qPCR

• Compare enrichment to input DNA and IgG control

Based on studies with similar subtelomeric genes, design primers around the SPAC186.06 locus and regions where binding is expected .

What controls should be included when working with SPAC186.06 Antibody?

Essential Controls for SPAC186.06 Antibody Experiments:

How can SPAC186.06 Antibody be used to study subtelomeric gene regulation in fission yeast?

SPAC186.06 is located in the subtelomeric region of chromosomes, which makes it valuable for studying unique regulatory mechanisms. Based on research with similar genes:

Research Strategy:

• Chromatin State Analysis: Use ChIP with SPAC186.06 Antibody alongside anti-Swi6 antibodies to assess correlation between heterochromatin formation and SPAC186.06 protein localization. Based on findings with related genes, the binding of Swi6 to subtelomeric regions extends approximately 50-90kb from chromosome ends .

• Epigenetic Modifications: Combine SPAC186.06 ChIP data with ChIP for histone modifications such as H3K4Me3, H3K9Ac, and H4K12Ac to map regulatory landscapes .

• Aneuploid Analysis: Compare SPAC186.06 expression and localization in normal haploid and aneuploid strains. Studies have shown that genes in similar regions exhibit significantly altered expression in aneuploids containing additional chromosomes (such as Ch10 or Ch16) .

• Heterochromatin Boundary Function: Investigate if SPAC186.06 plays a role in establishing or maintaining heterochromatin boundaries, similar to other subtelomeric proteins.

What is the relationship between SPAC186.06 and cytoplasmic freezing in S. pombe?

Research on cytoplasmic freezing (CF) in S. pombe has revealed complex cellular responses to starvation conditions. While direct evidence for SPAC186.06 involvement in CF isn't explicitly stated in the search results, its location and expression patterns suggest potential connections:

Investigative Approach:

• Starvation Response: Monitor SPAC186.06 expression and protein levels during CF induction using the standard protocol:

  • Grow cells in appropriate medium

  • Transfer to starvation conditions

  • Analyze protein levels via Western blotting with SPAC186.06 Antibody at different time points

• Quiescence State Analysis: Determine if SPAC186.06 is differentially expressed in the preparatory phase prior to CF induction, as research indicates "de novo production of components required for CF" during this phase .

• CF Phenotype in Gene Deletion: Create and analyze Δspac186.06 deletion strains to assess:

  • CF induction efficiency

  • Cytoplasmic solidification using methods such as:

    • Single particle tracking of lipid droplets

    • Mean squared displacement (MSD) of mitochondria

    • Bodipy correlation coefficient measurements

• Transcriptional Analysis: Perform real-time quantitative PCR (qRT-PCR) to analyze SPAC186.06 expression during:

  • Standard growth

  • Early starvation

  • Established quiescence

  • During CF induction and maintenance

The methodology would follow established protocols: RNA extraction using hot phenol method, DNase treatment, reverse transcription with ImProm-II Reverse Transcriptase, and qPCR with appropriate controls .

How does SPAC186.06 relate to TORC2-Gad8 signaling pathway in S. pombe?

The TORC2-Gad8 signaling pathway plays important roles in regulating gene expression in S. pombe. While direct evidence for SPAC186.06 involvement isn't explicitly stated, its subtelomeric location suggests potential regulation by this pathway:

Research Strategy:

• Protein Interaction Studies: Use co-immunoprecipitation (co-IP) to investigate potential physical interactions:

  • Prepare protein extracts from S. pombe expressing tagged Gad8

  • Perform IP using SPAC186.06 Antibody

  • Analyze precipitates for the presence of Gad8 by Western blotting

  • Conversely, perform IP with anti-Gad8 antibodies and probe for SPAC186.06

• Transcriptional Regulation Analysis: Compare SPAC186.06 expression in:

  • Wild-type strains

  • Δtor1 mutant cells

  • Gad8 phosphorylation site mutants

• Chromatin Association: Investigate if Gad8 associates with the SPAC186.06 promoter through ChIP:

  • Perform ChIP with anti-Gad8 antibodies

  • Design primers specific to the SPAC186.06 promoter region

  • Quantify enrichment via qPCR

• Stress Response Correlation: Determine if SPAC186.06 is among the stress-response genes regulated by TORC2-Gad8 signaling by examining expression changes under various stress conditions .

How can I validate the specificity of SPAC186.06 Antibody?

Comprehensive Validation Strategy:

• Western Blot Analysis:

  • Compare protein detection in wild-type vs. Δspac186.06 deletion strains

  • Verify a single band of expected molecular weight

  • Perform peptide competition assay by pre-incubating antibody with recombinant SPAC186.06 protein

• Cross-Reactivity Assessment:

  • Test antibody against recombinant proteins of related genes (SPAC186.05c, SPAC186.07c)

  • Examine reactivity in epitope-tagged strains (e.g., SPAC186.06-HA)

• Mass Spectrometry Validation:

  • Perform immunoprecipitation with SPAC186.06 Antibody

  • Analyze precipitated proteins by mass spectrometry

  • Confirm presence of SPAC186.06 peptides

• Functional Validation:

  • Compare protein localization in wild-type vs. conditional expression strains

  • Correlate antibody signal intensity with mRNA expression levels measured by RT-qPCR

What are common issues when using SPAC186.06 Antibody in immunofluorescence and how can they be addressed?

While specific immunofluorescence (IF) protocols for SPAC186.06 Antibody are not explicitly provided in the search results, the following general guidance can be extrapolated based on experience with similar fission yeast antibodies:

Common Issues and Solutions:

Optimized IF Protocol for S. pombe:

• Grow cells to mid-log phase in appropriate medium

• Fix with 3% formaldehyde for 30 minutes at 18°C

• Quench with 0.125M glycine and wash with PBS

• Digest cell wall with Zymolyase in buffer containing 1.2M sorbitol

• Permeabilize with 1% Triton X-100

• Block with 5% BSA in PBS

• Incubate with SPAC186.06 Antibody (1:100 dilution) overnight at 4°C

• Wash extensively with PBS + 0.1% Tween-20

• Incubate with fluorescent secondary antibody (e.g., anti-rabbit IgG conjugated to Alexa Fluor 488)

• Counterstain nuclei with DAPI

• Mount and image using confocal microscopy

What are the key considerations for quantitative analysis of SPAC186.06 protein levels?

For accurate quantification of SPAC186.06 protein levels:

Quantitative Western Blot:

• Sample Preparation Consistency:

  • Standardize cell lysis protocol

  • Determine protein concentration by Bradford or BCA assay

  • Load equal amounts of protein (15-20μg) per lane

• Controls and Normalization:

  • Include loading control (actin) in each blot

  • Use recombinant SPAC186.06 protein to create a standard curve

  • Include the same positive control sample across all blots for inter-blot normalization

• Detection and Quantification:

  • Use digital imaging systems rather than film for wider dynamic range

  • Ensure signal is within linear range of detection

  • Quantify band intensity using software like ImageJ

  • Calculate relative expression as: (SPAC186.06 signal / loading control signal)

• Statistical Analysis:

  • Perform experiments in biological triplicates

  • Apply appropriate statistical tests (t-test or ANOVA)

  • Report data as mean ± standard deviation

ELISA-Based Quantification:

• Consider developing a sandwich ELISA using SPAC186.06 Antibody as capture antibody

• Use appropriate detection systems following manufacturers' protocols

• Include standard curves with recombinant protein

How does genomic location affect SPAC186.06 expression and function?

Research on similar subtelomeric genes provides insights into how genomic location might affect SPAC186.06:

Current Understanding:

• Heterochromatin Regulation: SPAC186.06 is located in regions typically bound by Swi6, suggesting its expression may be regulated by heterochromatin formation .

• Telomere Position Effect: Based on studies of related genes, the proximity to telomeres likely influences SPAC186.06 expression through telomere position effect (TPE).

• Dosage Sensitivity: Analysis of aneuploid strains has shown that related genes (SPAC186.05c, SPAC186.04c) exhibit significant expression changes when chromosome copy numbers are altered .

Research Approaches:

• Positional Transplantation: Clone SPAC186.06 to different genomic locations and measure expression changes

• Boundary Element Analysis: Investigate if SPAC186.06 locus contains boundary elements that separate heterochromatin from euchromatin

• Chromatin Structure Mapping: Perform high-resolution chromatin analysis around the SPAC186.06 locus using techniques like Micro-C or Hi-C

• Long-Range Interaction Studies: Investigate potential interactions between SPAC186.06 locus and distant genomic regions

What is the role of SPAC186.06 in the metabolic adaptation of S. pombe?

While specific metabolic functions of SPAC186.06 aren't explicitly described in the search results, its potential role can be investigated based on approaches used for similar genes:

Investigative Methodology:

• Metabolic Profiling:

  • Compare metabolite profiles between wild-type and Δspac186.06 strains

  • Analyze changes under different carbon sources (glucose, glycerol, acetate)

  • Apply 13C-based metabolic flux analysis to trace carbon flow

• Growth Condition Response:

  • Monitor SPAC186.06 expression during:

    • Fermentative growth

    • Respiratory growth

    • Nutrient limitation

    • Stationary phase

• Protein-Protein Interactions:

  • Use SPAC186.06 Antibody for immunoprecipitation followed by mass spectrometry

  • Identify potential metabolic enzymes or regulators that interact with SPAC186.06

• Phenotypic Analysis:

  • Assess growth rates of Δspac186.06 strains under various metabolic conditions

  • Measure key metabolic parameters (oxygen consumption, fermentation products)

  • Evaluate stress resistance phenotypes

By integrating these approaches, researchers can develop a comprehensive understanding of SPAC186.06's function and its potential applications in both basic and applied research.