SPAC186.04c Antibody

What is SPAC186.04c and why is it significant for S. pombe research?

SPAC186.04c (UniProt ID: G2TRL1) is a gene encoding a protein in Schizosaccharomyces pombe (strain 972/ATCC 24843), commonly known as fission yeast . This protein has been studied in the context of gene expression analyses and chromosome function. Research involving SPAC186.04c can provide insights into fundamental cellular processes in S. pombe, which serves as an important model organism for eukaryotic cell biology.

The significance of studying this protein lies in understanding its potential role in various cellular pathways. Like other S. pombe genes that have been characterized, SPAC186.04c may participate in processes such as cell cycle regulation, stress response, or chromosome dynamics, as observed with other genes in similar studies .

What validation methods should be employed to confirm SPAC186.04c antibody specificity?

Antibody validation is critical for ensuring experimental reproducibility. For SPAC186.04c antibody, employ these methodological approaches:

• Western blot analysis with positive and negative controls:

  • Use wild-type S. pombe extracts as positive control

  • Include knockout or knockdown samples as negative controls

  • Assess band specificity at the predicted molecular weight

• Pre-absorption test:

  • Incubate the antibody with recombinant SPAC186.04c protein

  • Perform parallel Western blots with treated and untreated antibody

  • Loss of signal in pre-absorbed samples confirms specificity

• Cross-reactivity assessment:

  • Test against related proteins or in heterologous expression systems

  • Evaluate detection in non-target species

• Immunoprecipitation followed by mass spectrometry:

  • Perform IP with the antibody and analyze pulled-down proteins

  • Confirm predominant enrichment of SPAC186.04c

Similar validation techniques have been successfully applied to antibodies against other S. pombe proteins, such as Rhb1 GTPase, which was validated using recombinant protein adsorption tests .

What are the optimal experimental conditions for Western blotting with SPAC186.04c antibody?

Based on standardized protocols for S. pombe proteins, the following methodological guidelines are recommended:

Sample preparation:

• Harvest cells at 1 × 10^7 cells/ml concentration

• Prepare extracts in buffer containing phosphate buffer (PBS), 1 mM MgCl₂, 0.5% Triton X-100, and 0.5% deoxycholate

• Add protease inhibitors: 1 mM PMSF and 1× protease inhibitor cocktail

Gel electrophoresis and transfer:

• Load equal amounts of protein (15-20 μg) on 15% polyacrylamide gels

• Transfer to nitrocellulose membranes at 100V for 1 hour

Antibody incubation:

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

• Incubate with SPAC186.04c antibody at 1:2000 dilution (adjust based on titer)

• Incubate overnight at 4°C

• Wash 3× with TBST

• Incubate with appropriate secondary antibody (HRP-conjugated)

Detection and normalization:

• Use ECL substrate for detection

• Include anti-α-tubulin antibody (such as TAT-1) as loading control

How can SPAC186.04c antibody be optimized for chromatin immunoprecipitation (ChIP) experiments?

ChIP optimization for SPAC186.04c antibody follows this methodological framework:

Chromatin preparation protocol:

• Fix exponentially growing S. pombe cells (5×10^8) with 3% formaldehyde in YES medium for 30 minutes at 18°C

• Add glycine to 0.125 M final concentration to quench fixation

• Wash cells twice with ice-cold PBS

• Lyse cells and sonicate chromatin to generate fragments (200-500 bp)

Immunoprecipitation procedure:

• Pre-clear chromatin with protein A agarose beads

• Incubate chromatin with SPAC186.04c antibody for 4 hours at 4°C

• Add protein A agarose beads and incubate for 1 hour at 4°C

• Precipitate beads by centrifugation at 5000 rpm for 1 minute

• Wash extensively and elute bound material

Analysis methods:

• Reverse crosslinks and purify DNA

• Analyze by qPCR or next-generation sequencing

• Compare enrichment to input control and IgG control

This protocol has been successfully applied to chromatin proteins such as Swi6 in S. pombe, which demonstrated specific binding patterns at subtelomeric regions and centromeres .

What approaches should be used to troubleshoot weak or non-specific signals when using SPAC186.04c antibody?

When encountering issues with SPAC186.04c antibody performance, implement this systematic troubleshooting approach:

For S. pombe proteins, specific considerations include possible modification states affecting antibody recognition, as observed with other proteins like Rhb1, which shows different migration patterns depending on its farnesylation status .

How can SPAC186.04c antibody be effectively used for immunoprecipitation to study protein-protein interactions?

To optimize immunoprecipitation with SPAC186.04c antibody, follow this methodological workflow:

Lysis buffer optimization:

• Use buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100

• Add phosphatase inhibitors if studying phosphorylation events

• Include 1 mM DTT if protein contains disulfide bonds

Immunoprecipitation protocol:

• Prepare cell lysates from 5×10^8 S. pombe cells

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

• Incubate cleared lysate with SPAC186.04c antibody (2-5 μg) overnight at 4°C

• Add protein A/G beads and incubate for 2-4 hours

• Wash beads 4-5 times with lysis buffer

• Elute bound proteins with SDS sample buffer or by competition with peptide

Verification methods:

• Analyze by Western blot with antibodies against suspected interaction partners

• Perform reverse IP with antibodies against putative partners

• Consider mass spectrometry to identify novel interactions

This approach has been successfully used to characterize protein interactions for other S. pombe proteins, enabling identification of specific binding partners and functional complexes .

What methods can be used to study SPAC186.04c expression patterns across different genetic backgrounds?

To analyze SPAC186.04c expression patterns, implement these methodological approaches:

Quantitative transcript analysis:

• Extract total RNA from different S. pombe strains

• Perform qRT-PCR with SPAC186.04c-specific primers

• Normalize to appropriate reference genes (e.g., act1+, cdc2+)

• Calculate relative expression using ΔΔCt method

Microarray analysis:

• Prepare labeled cDNA from total RNA

• Hybridize to S. pombe-specific microarrays

• Consider a significant change when r' > 2 or r' < -2

Protein level analysis:

• Perform Western blot with SPAC186.04c antibody

• Quantify band intensities using densitometry

• Normalize to loading controls

These methods have been applied to study gene expression changes in S. pombe partial aneuploids, where significant expression changes were documented for genes in altered chromosomal regions .

How does SPAC186.04c expression compare between wild-type and partial aneuploid S. pombe strains?

While specific data for SPAC186.04c is not directly presented in the search results, similar genes have shown characteristic expression patterns in aneuploid strains that can inform research approaches:

• Genes located on duplicated chromosomal regions typically show approximately 1.5 to 2-fold increased expression in partial aneuploids compared to wild-type strains

• Expression changes may be influenced by chromosomal location, particularly proximity to telomeres or centromeres where chromatin structure affects gene expression

• Comparative expression analysis between normal haploid and aneuploid strains can reveal:

  • Direct gene dosage effects

  • Secondary changes in gene regulation

  • Compensatory expression mechanisms

Similar studies with other S. pombe genes have shown that genes located near telomeres may exhibit altered expression patterns in aneuploid strains, possibly due to changes in the binding of heterochromatin proteins like Swi6 .

How can ChIP-seq data be analyzed to understand SPAC186.04c chromatin associations?

For analyzing ChIP-seq data to investigate SPAC186.04c chromatin associations, implement this analytical framework:

Data processing pipeline:

• Align sequencing reads to S. pombe reference genome

• Remove PCR duplicates and filter for quality

• Call peaks using MACS2 or similar algorithm

• Compare enrichment to input control and IgG control

Visualization and interpretation:

• Generate genome browser tracks showing binding profiles

• Create heatmaps of binding at relevant genomic features

• Perform motif enrichment analysis for binding sites

• Integrate with gene expression data

Comparative analysis approach:

• Compare SPAC186.04c binding profiles between different conditions

• Correlate binding with changes in gene expression

• Analyze co-occupancy with other chromatin factors

This analytical approach has been effectively applied to chromatin proteins in S. pombe, such as Swi6, which showed specific binding patterns at heterochromatic regions including centromeres and subtelomeres .

What considerations should be made when developing custom antibodies against SPAC186.04c?

When developing custom antibodies against SPAC186.04c, implement these methodological considerations:

Antigen design strategies:

• Recombinant protein approach:

  • Express full-length SPAC186.04c with His-tag in E. coli

  • Purify using affinity chromatography (e.g., MagneHis system)

  • Use for immunization of rabbits

• Peptide-based approach:

  • Identify antigenic regions using epitope prediction tools

  • Select peptides of 15-20 amino acids with high antigenicity

  • Conjugate to carrier protein (KLH or BSA)

  • Use for immunization

Production and purification:

• Generate polyclonal antibodies in rabbits using standard protocols

• Consider monoclonal antibody production for higher specificity

• Purify antibodies using protein A/G affinity chromatography

• Perform affinity purification against immobilized antigen

Validation requirements:

• Test antibody specificity against recombinant SPAC186.04c

• Verify recognition of endogenous protein in S. pombe extracts

• Perform pre-absorption tests to confirm specificity

• Validate in multiple applications (Western blot, IP, IF)

This approach has been successfully used to generate antibodies against other S. pombe proteins, such as Rhb1, which were subsequently validated for specificity and used in various applications .

How can SPAC186.04c antibody be used to study protein modifications and their functional significance?

To investigate protein modifications of SPAC186.04c, implement these methodological approaches:

Detection of post-translational modifications:

• Phosphorylation analysis:

  • Treat samples with lambda phosphatase

  • Compare migration patterns before and after treatment

  • Use phospho-specific antibodies if phosphorylation sites are known

• Ubiquitination/SUMOylation assessment:

  • Perform immunoprecipitation under denaturing conditions

  • Probe with anti-ubiquitin or anti-SUMO antibodies

  • Use proteasome inhibitors to stabilize modified forms

• Farnesylation or other lipid modifications:

  • Analyze mobility shifts on SDS-PAGE

  • Compare cytosolic and membrane fractions

  • Use specific inhibitors of modification pathways

Functional significance analysis:

• Study modification changes under different conditions

• Correlate modifications with protein localization

• Generate mutants that cannot be modified

• Assess phenotypic consequences

This approach has revealed important insights for other S. pombe proteins, such as Rhb1 GTPase, where farnesylation was shown to be critical for proper membrane association and function, with the unmodified form showing different migration patterns on SDS-PAGE .

What experimental design principles should be followed when using SPAC186.04c antibody in comparative proteomic studies?

For robust comparative proteomic studies using SPAC186.04c antibody, implement these experimental design principles:

Sample preparation considerations:

• Process all samples in parallel to minimize batch effects

• Include biological replicates (minimum n=3) for statistical power

• Standardize cell growth conditions and harvesting procedures

• Use SILAC or TMT labeling for quantitative comparisons

Experimental controls:

• Include wild-type, knockout, and overexpression samples

• Add IgG control for immunoprecipitation experiments

• Incorporate spike-in standards for quantification

• Use reciprocal labeling in SILAC experiments

Analytical workflow:

• Perform immunoprecipitation with SPAC186.04c antibody

• Analyze by LC-MS/MS with high resolution

• Identify proteins using database search algorithms

• Quantify using label-free or labeled approaches

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

Data interpretation framework:

• Filter results based on fold change and statistical significance

• Classify interactions as stable or transient

• Perform GO term and pathway enrichment analysis

• Validate key findings using orthogonal methods

This methodological approach has been successfully applied in studies of other S. pombe proteins, enabling identification of specific protein interactions and their functional significance .

How can SPAC186.04c antibody be integrated with genomic data to understand its role in chromosome dynamics?

To integrate SPAC186.04c antibody studies with genomic approaches, implement this multidisciplinary framework:

Integrated experimental design:

• Perform ChIP-seq with SPAC186.04c antibody across different conditions

• Generate RNA-seq data from the same conditions

• Collect proteomics data using IP-MS approaches

• Integrate with publicly available genomic datasets

Data integration methods:

• Correlate SPAC186.04c binding sites with gene expression changes

• Map binding patterns relative to chromosome landmarks

• Compare binding profiles with histone modifications

• Integrate with chromosome conformation capture data (Hi-C)

Analytical approaches:

• Use machine learning to identify patterns in binding data

• Develop network models incorporating multiple data types

• Apply statistical methods to identify significant associations

• Visualize integrated data using genome browsers and heatmaps

This integrative approach has been applied to study chromosome dynamics in S. pombe, revealing important insights into heterochromatin formation and the role of proteins like Swi6 in maintaining chromosome structure .

What methodologies should be used to study the relationship between SPAC186.04c and heterochromatin proteins like Swi6?

To investigate potential functional relationships between SPAC186.04c and heterochromatin proteins, implement these methodological approaches:

Co-localization studies:

• Perform sequential ChIP (re-ChIP) to assess co-occupancy

• Compare binding profiles from individual ChIP experiments

• Conduct co-immunofluorescence microscopy

• Analyze protein proximity using PLA or FRET

Genetic interaction analysis:

• Create single and double mutants/deletions

• Assess phenotypic consequences using growth assays

• Measure gene expression changes using RNA-seq

• Evaluate heterochromatin stability using reporter assays

Biochemical interaction studies:

• Perform co-immunoprecipitation experiments

• Use yeast two-hybrid or BioID approaches

• Conduct in vitro binding assays with purified proteins

• Apply protein crosslinking followed by mass spectrometry

Similar studies with Swi6 in S. pombe have revealed its critical role in heterochromatin formation at centromeres and telomeres, with specific binding patterns that are altered in aneuploid strains .

This type of analysis could reveal whether SPAC186.04c shares binding regions with heterochromatin proteins or shows distinct localization patterns, providing insights into its potential function.

How can CRISPR-based technologies be combined with SPAC186.04c antibody for functional genomics studies?

To leverage CRISPR technologies with SPAC186.04c antibody studies, implement these integrated approaches:

CRISPR-based genomic modifications:

• Generate SPAC186.04c knockout using CRISPR-Cas9

• Create endogenous tags for visualization and purification

• Introduce specific mutations to study functional domains

• Develop inducible degradation systems

CUT&RUN or CUT&Tag alternatives to traditional ChIP:

• Use SPAC186.04c antibody with Protein A-MNase fusion

• Target DNA cleavage specifically at binding sites

• Sequence released fragments for high-resolution mapping

• Achieve higher signal-to-noise ratio than conventional ChIP

CRISPR activation/repression systems:

• Target SPAC186.04c locus with CRISPRa or CRISPRi

• Measure effects on gene expression and cellular phenotypes

• Use SPAC186.04c antibody to confirm protein level changes

• Identify downstream effects through proteomics

These methodologies represent cutting-edge approaches that can be applied to study SPAC186.04c function, similar to advanced studies performed on other S. pombe proteins .

What considerations should be made when using SPAC186.04c antibody in multiplexed immunoassays?

For implementing multiplexed immunoassays with SPAC186.04c antibody, consider these methodological guidelines:

Antibody compatibility assessment:

• Test for cross-reactivity between multiple antibodies

• Verify epitope accessibility in multiplexed formats

• Validate detection systems for specificity

• Establish optimal antibody concentrations

Multiplexed detection platforms:

• Microarray-based approaches:

  • Print capture antibodies in defined patterns

  • Incubate with complex samples

  • Detect using fluorescently labeled antibodies

• Bead-based systems:

  • Conjugate SPAC186.04c antibody to uniquely coded beads

  • Combine with beads carrying other antibodies

  • Analyze using flow cytometry

• Sequential elution techniques:

  • Apply antibodies in sequence with elution steps

  • Image after each antibody application

  • Compile data from sequential rounds

Data normalization and analysis:

• Include internal standards for quantification

• Apply appropriate statistical methods for multiplexed data

• Account for potential cross-talk between detection channels

• Use machine learning for pattern recognition

These approaches have been successfully applied in antibody characterization platforms, enabling simultaneous evaluation of multiple parameters in complex biological samples .

How might nanobody-based approaches complement traditional SPAC186.04c antibody applications?

Nanobody-based approaches offer several methodological advantages that can complement traditional antibody applications for SPAC186.04c:

Nanobody development pipeline:

• Immunize camelids with recombinant SPAC186.04c

• Clone VHH domains from B cells

• Select specific binders through phage display

• Express and purify recombinant nanobodies

Comparative advantages for specific applications:

• Structural biology:

  • Smaller size (15 kDa vs. 150 kDa) allows better crystal packing

  • Can access epitopes in protein cavities

  • Stabilize specific protein conformations

• Live-cell imaging:

  • Better tissue penetration

  • Expression as intrabodies for real-time dynamics

  • Lower background in cellular environments

• High-resolution microscopy:

  • Reduced linkage error in super-resolution techniques

  • Improved localization precision

  • Higher labeling density

Potential applications for SPAC186.04c studies:

• Tracking protein dynamics in living S. pombe cells

• Capturing transient protein-protein interactions

• Developing affinity purification approaches with reduced background

• Creating biosensors for protein modifications

These nanobody-based approaches represent cutting-edge methodologies that could advance understanding of SPAC186.04c function in ways complementary to traditional antibody applications .