SPAC977.08 Antibody

What is the SPAC977.08 antibody and what are its basic characteristics?

SPAC977.08 antibody is a polyclonal antibody raised in rabbits against recombinant Schizosaccharomyces pombe (strain 972 / ATCC 24843) SPAC977.08 protein. The antibody is antigen affinity purified and formulated in a storage buffer containing 0.03% Proclin 300 as a preservative, 50% glycerol, and 0.01M PBS at pH 7.4. It is specifically designed for detection of the S. pombe SPAC977.08 gene product in research applications .

What are the validated applications for SPAC977.08 antibody?

The SPAC977.08 antibody has been validated for enzyme-linked immunosorbent assay (ELISA) and Western blotting (WB) applications. The antibody is provided as a non-conjugated polyclonal preparation at a concentration suitable for direct use in these applications. Researchers should conduct preliminary validation experiments to determine optimal working dilutions for their specific experimental conditions .

What are the optimal storage conditions for SPAC977.08 antibody?

For maximum stability and activity retention, SPAC977.08 antibody should be stored at -20°C or -80°C upon receipt. The antibody is supplied in a formulation containing 50% glycerol, which prevents freezing at -20°C and helps maintain antibody activity. Repeated freeze-thaw cycles should be avoided as they can lead to protein denaturation and loss of antibody function. For short-term storage (less than one month), the antibody can be kept at 2-8°C , similar to other research antibodies .

What are the recommended protocols for using SPAC977.08 antibody in Western blotting?

For optimal Western blotting results with SPAC977.08 antibody:

• Sample preparation: Prepare S. pombe cell lysates in a denaturing buffer containing protease inhibitors

• Protein separation: Use SDS-PAGE with appropriate percentage acrylamide gels (10-12% recommended)

• Transfer: Transfer proteins to PVDF or nitrocellulose membranes using standard protocols

• Blocking: Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Primary antibody: Dilute SPAC977.08 antibody in blocking buffer (starting dilution 1:1000) and incubate overnight at 4°C

• Washing: Wash 3-5 times with TBST

• Secondary antibody: Use appropriate anti-rabbit IgG secondary antibody conjugated to HRP

• Detection: Visualize using chemiluminescence substrate

For comprehensive controls, compare with negative controls (lysates from deletion strains) and positive controls (overexpression strains if available) .

How should S. pombe samples be prepared for optimal antibody reactivity?

For optimal antibody detection of SPAC977.08 protein:

Cell lysis protocol for S. pombe:

• Grow S. pombe cultures to mid-log phase (OD600 0.5-0.8)

• Harvest cells by centrifugation (3,000g for 5 min)

• Wash cell pellet with cold PBS

• Resuspend cells in lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10% glycerol, protease inhibitor cocktail)

• Add glass beads (0.5 mm) to the cell suspension (1:1 ratio)

• Lyse cells using a bead beater (8 cycles of 30 seconds beating/30 seconds on ice)

• Centrifuge at 15,000g for 15 minutes at 4°C

• Collect supernatant for antibody applications

This method ensures efficient extraction of SPAC977.08 protein while preserving antibody epitopes. For membrane-associated proteins, consider additional detergent optimization steps .

How can SPAC977.08 antibody be used in chromatin immunoprecipitation (ChIP) experiments?

Although not explicitly validated for ChIP, researchers interested in adapting SPAC977.08 antibody for chromatin immunoprecipitation can implement the following protocol:

ChIP Protocol for S. pombe:

• Cross-link S. pombe cells with 1% formaldehyde for 15 minutes at room temperature

• Quench with 125 mM glycine for 5 minutes

• Harvest cells and wash with cold PBS

• Resuspend in lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, protease inhibitors)

• Lyse cells using glass beads

• Sonicate chromatin to 200-500 bp fragments

• Pre-clear lysate with Protein A/G beads

• Incubate with SPAC977.08 antibody (5-10 μg per sample) overnight at 4°C

• Add Protein A/G beads and incubate for 2-3 hours

• Wash beads with increasingly stringent buffers

• Elute DNA-protein complexes and reverse cross-links

• Purify DNA for downstream analysis

Include appropriate controls: input chromatin, non-specific IgG antibody control, and positive control regions .

How can SPAC977.08 antibody be used with CRISPR genome editing in S. pombe to study protein function?

Researchers can employ CRISPR-Cas9 genome editing in conjunction with SPAC977.08 antibody to study protein function using this comprehensive approach:

• Epitope tagging strategy:

  • Design CRISPR-Cas9 construct targeting the C-terminus of SPAC977.08

  • Create repair template with HA or FLAG tag sequence

  • Transform S. pombe with CRISPR construct and repair template

  • Confirm successful tagging by PCR and sequencing

• Functional validation:

  • Use SPAC977.08 antibody to verify expression of the native protein

  • Use anti-tag antibody to verify expression of the tagged protein

  • Compare localization and expression levels between native and tagged versions

• Phenotypic analysis:

  • Create SPAC977.08 knockout using CRISPR-Cas9

  • Compare protein absence using SPAC977.08 antibody

  • Assess phenotypic changes (growth rate, morphology, stress response)

  • Perform rescue experiments with wild-type or mutant constructs

This approach combines genetic manipulation with antibody-based validation to provide comprehensive insights into SPAC977.08 function .

How should Western blot data using SPAC977.08 antibody be quantified and normalized?

For rigorous quantification of Western blot data using SPAC977.08 antibody:

• Image acquisition:

  • Use a digital imaging system with a wide dynamic range

  • Capture images before signal saturation

  • Obtain multiple exposures if necessary

• Quantification method:

  • Measure band intensity using software like ImageJ or dedicated Western blot analysis software

  • Subtract local background from each band

  • Plot intensity values against a standard curve if absolute quantification is required

• Normalization approach:

  • Normalize to appropriate loading controls (e.g., α-tubulin, GAPDH, or total protein stain)

  • For S. pombe, consider Cdc2 or Act1 as stable reference proteins

  • Calculate relative expression as: (Target protein intensity/Loading control intensity)

• Statistical analysis:

  • Perform experiments in biological triplicates

  • Apply appropriate statistical tests (t-test for two conditions, ANOVA for multiple conditions)

  • Present data as mean ± standard deviation or standard error

This approach ensures reproducible and reliable quantification of SPAC977.08 protein levels .

How can researchers verify the specificity of SPAC977.08 antibody in their experimental system?

To rigorously verify SPAC977.08 antibody specificity:

Comprehensive validation protocol:

• Genetic controls:

  • Test antibody on samples from SPAC977.08 deletion strain (negative control)

  • Test on samples from SPAC977.08 overexpression strain (positive control)

  • If available, test on samples expressing tagged versions of SPAC977.08

• Biochemical validation:

  • Perform peptide competition assay by pre-incubating antibody with excess immunizing peptide

  • Verify single band of expected molecular weight in Western blot

  • Confirm absence of non-specific bands in knockout/deletion samples

• Cross-reactivity assessment:

  • Test antibody on related S. pombe proteins if sequence homology exists

  • Test on proteins from other yeast species (S. cerevisiae) to assess cross-species reactivity

  • Evaluate potential cross-reactivity with human proteins if translational research is planned

• Technical validation:

  • Compare results across multiple antibody dilutions

  • Test different blocking reagents to optimize signal-to-noise ratio

  • Compare fresh versus frozen samples to assess epitope stability

These comprehensive validation steps ensure antibody specificity and experimental reproducibility .

What are common issues when using SPAC977.08 antibody in immunoprecipitation and how can they be resolved?

While SPAC977.08 antibody isn't explicitly validated for immunoprecipitation, researchers may adapt it for this application. Common issues and solutions include:

When adapting this antibody for IP applications, thorough validation and optimization are necessary .

How can SPAC977.08 antibody be used in conjunction with transcriptomics data to understand gene regulation in S. pombe?

Researchers can integrate SPAC977.08 antibody studies with transcriptomics to understand gene regulation through this comprehensive approach:

• Integrative experimental design:

  • Perform RNA-seq under conditions of interest (stress, cell cycle phases, etc.)

  • Simultaneously collect protein samples for SPAC977.08 antibody detection

  • Consider ChIP-seq if SPAC977.08 is suspected to interact with chromatin

• Data integration methodology:

  • Analyze differential gene expression from RNA-seq data

  • Quantify SPAC977.08 protein levels using the antibody in Western blots

  • Correlate protein expression with transcriptional changes

  • Identify genes showing coordinated regulation with SPAC977.08

• Functional validation:

  • For genes showing correlation, perform genetic interaction studies

  • Use CRISPR interference to modulate SPAC977.08 and measure effects on target genes

  • Confirm protein-level changes using antibodies against putative targets

This integrated approach bridges transcriptional data with protein-level analyses to elucidate SPAC977.08's role in gene regulation networks .

How can SPAC977.08 antibody be used to study heterochromatin formation in S. pombe?

Given S. pombe's importance as a model for studying heterochromatin, SPAC977.08 antibody can be incorporated into heterochromatin research using this methodology:

• Co-localization studies:

  • Perform immunofluorescence with SPAC977.08 antibody alongside established heterochromatin markers (e.g., anti-H3K9me, Swi6)

  • Quantify co-localization using confocal microscopy and correlation analysis

  • Assess changes in localization patterns under different conditions

• Chromatin association analysis:

  • Conduct ChIP-seq with SPAC977.08 antibody to map genomic binding sites

  • Compare binding profiles with known heterochromatin regions (centromeres, telomeres, mating loci)

  • Analyze binding in wild-type versus heterochromatin mutants (clr4Δ, swi6Δ)

• Functional relationship assessment:

  • Generate SPAC977.08 deletion strains and analyze heterochromatin integrity

  • Measure silencing of reporter genes integrated at heterochromatic loci

  • Assess H3K9 methylation levels in SPAC977.08 mutants

This comprehensive approach can determine if SPAC977.08 contributes to heterochromatin formation or maintenance in S. pombe .

What approaches can be used to adapt SPAC977.08 antibody for flow cytometry applications?

Although not specifically validated for flow cytometry, researchers can adapt SPAC977.08 antibody for this application using this systematic approach:

• Cell preparation protocol:

  • Harvest S. pombe cells in log phase

  • Fix with 4% paraformaldehyde for 15 minutes

  • Permeabilize with 0.1% Triton X-100 for 10 minutes

  • Block with 3% BSA in PBS for 30 minutes

  • Incubate with SPAC977.08 antibody (1:100-1:500 dilution)

  • Wash with PBS

  • Incubate with fluorophore-conjugated anti-rabbit secondary antibody

• Optimization parameters:

  • Test multiple fixation/permeabilization protocols

  • Titrate primary and secondary antibody concentrations

  • Compare intracellular versus surface staining protocols

  • Include appropriate isotype controls

• Validation experiments:

  • Compare wild-type versus deletion strains

  • Use GFP-tagged SPAC977.08 expressing strains as positive controls

  • Perform competitive binding with immunizing peptide

• Analysis considerations:

  • Gate on singlet populations

  • Compare fluorescence intensity distributions across conditions

  • Consider dual staining with cell cycle markers (DNA content)

These steps provide a methodical approach to adapting SPAC977.08 antibody for flow cytometry applications .

How can SPAC977.08 antibody be incorporated into large-scale proteomic studies of S. pombe?

SPAC977.08 antibody can be integrated into comprehensive proteomic workflows using the following methodology:

• Immunoaffinity enrichment strategy:

  • Conjugate SPAC977.08 antibody to agarose or magnetic beads

  • Use for affinity purification of protein complexes

  • Elute and analyze by LC-MS/MS to identify interaction partners

  • Compare results with BioGRID or PomBase interaction databases

• Targeted proteomics approach:

  • Develop multiple reaction monitoring (MRM) assays for SPAC977.08 peptides

  • Use antibody-based enrichment to increase detection sensitivity

  • Quantify SPAC977.08 across different conditions or genetic backgrounds

  • Correlate with other measured proteins to identify functional relationships

• Spatial proteomics integration:

  • Use fractionation to isolate subcellular compartments

  • Apply SPAC977.08 antibody to detect localization across fractions

  • Combine with global proteomic profiling of the same fractions

  • Identify co-localizing proteins for functional hypotheses

This systematic approach leverages SPAC977.08 antibody for comprehensive proteomic investigations beyond simple detection .

What considerations are important when using SPAC977.08 antibody for comparative studies across different S. pombe strains?

When designing comparative studies using SPAC977.08 antibody across different S. pombe strains:

Experimental design considerations:

• Strain selection and verification:

  • Document complete genotypes of all strains

  • Verify genetic backgrounds by PCR or sequencing

  • Consider using isogenic strains differing only in the gene of interest

• Growth standardization:

  • Standardize culture conditions (media, temperature, growth phase)

  • Harvest cells at equivalent physiological states (OD600)

  • Process all samples in parallel to minimize technical variation

• Analysis controls:

  • Include common reference strain(s) across all experiments

  • Use loading controls specific for each subcellular compartment

  • Consider spike-in controls for absolute quantification

• Data normalization strategies:

  • Normalize to total protein rather than single housekeeping genes

  • Consider strain-specific variations in reference protein expression

  • Use multiple normalization approaches and compare results

• Statistical considerations:

  • Perform power analysis to determine required biological replicates

  • Apply appropriate statistical tests (consider strain-specific variances)

  • Use multiple comparison corrections for large strain panels