SPCC736.13 Antibody

What is SPCC736.13 and why is it significant in fission yeast research?

SPCC736.13 is a protein in Schizosaccharomyces pombe (fission yeast) identified by UniProt accession number O74959. While the search results don't specify its exact function, antibodies against this protein allow researchers to study its expression, localization, and interactions in fission yeast cellular processes. S. pombe is a model organism widely used to study fundamental cellular mechanisms including cell cycle regulation, DNA damage responses, and chromosome dynamics, making tools for studying specific proteins like SPCC736.13 valuable for basic research applications .

What are the primary validated applications for SPCC736.13 antibody?

Based on the available data, SPCC736.13 antibody has been validated for Western Blotting (WB) and ELISA applications. These methods allow researchers to detect and quantify the presence of SPCC736.13 protein in yeast cell lysates and other experimental samples. The antibody is specifically tested to ensure identification of the target antigen in these applications .

What are the optimal storage conditions for maintaining SPCC736.13 antibody activity?

The SPCC736.13 antibody should be stored at -20°C or -80°C upon receipt. Repeated freeze-thaw cycles should be avoided as they can degrade antibody quality and reduce binding efficiency. The antibody is supplied in liquid form containing 50% glycerol and 0.01M PBS (pH 7.4) with 0.03% Proclin 300 as a preservative, which helps maintain stability during storage .

How should researchers validate SPCC736.13 antibody specificity in their experimental systems?

Validation of SPCC736.13 antibody specificity should follow a multi-step approach:

• Positive and negative controls: Use wild-type S. pombe cells as positive controls and SPCC736.13 knockout strains (if available) as negative controls

• Peptide competition assay: Pre-incubate the antibody with excess recombinant SPCC736.13 protein before application to verify signal reduction

• Multiple detection methods: Confirm results using both ELISA and Western blot

• Molecular weight verification: Ensure the detected band in Western blots matches the expected molecular weight of SPCC736.13

• Cross-reactivity assessment: Test against closely related proteins to confirm specificity

What are the recommended dilution ranges for different experimental applications?

While specific dilution ranges aren't provided in the search results, researchers typically determine optimal working dilutions through titration experiments. For polyclonal antibodies like SPCC736.13 antibody:

• Western blotting: Start with 1:500 to 1:2000 dilutions

• ELISA: Begin with 1:1000 to 1:5000 dilutions

Optimization should involve testing multiple dilutions to identify the concentration that produces the strongest specific signal with minimal background. Document the batch-specific optimal conditions, as they may vary between antibody lots.

How can researchers optimize SPCC736.13 antibody performance in challenging experimental conditions?

When working with challenging experimental conditions:

• Sample preparation optimization:

  • For membrane proteins, test different lysis buffers with varying detergent compositions

  • Adjust protein extraction protocols to preserve epitope integrity

• Signal enhancement strategies:

  • Implement signal amplification systems like biotin-streptavidin

  • Use more sensitive detection substrates for Western blots

• Background reduction:

  • Increase blocking agent concentration (5% BSA or milk)

  • Include additional washing steps with increased salt concentration

  • Add 0.1-0.5% Tween-20 to reduce non-specific binding

• Cross-linking methods:

  • Consider DSS or formaldehyde cross-linking to capture transient protein interactions

These optimizations can significantly improve signal-to-noise ratio when working with low-abundance proteins or challenging sample types.

What considerations are important when designing co-immunoprecipitation experiments with SPCC736.13 antibody?

For successful co-immunoprecipitation (Co-IP) experiments:

• Buffer composition is critical:

  • Use buffers that maintain native protein conformation

  • Test different detergent concentrations (0.1-1% NP-40, Triton X-100, or CHAPS)

  • Include protease and phosphatase inhibitors to prevent degradation

• Antibody coupling:

  • Direct coupling to beads may reduce heavy chain interference in blotting

  • Consider crosslinking antibody to protein A/G beads using DMP or BS3

• Controls must include:

  • IgG control from the same species (rabbit)

  • Input sample (pre-immunoprecipitation)

  • Unrelated antibody control

  • If possible, SPCC736.13 knockout strain as negative control

• Elution conditions:

  • Test both harsh (SDS, low pH) and gentle (competitive peptide) elution methods

  • Consider native elution if downstream functional assays are planned

These considerations ensure reliable identification of genuine protein-protein interactions with minimal artifacts.

How can researchers distinguish between specific and non-specific signals when using SPCC736.13 antibody?

Distinguishing specific from non-specific signals requires systematic controls:

• Signal validation strategies:

  • Compare band patterns with predicted molecular weight of SPCC736.13

  • Use genetic knockdown/knockout controls when available

  • Perform peptide competition assays to block specific binding

• Signal pattern analysis:

  • Specific signals typically show consistent molecular weight across experiments

  • Non-specific signals often vary with experimental conditions

• Quantitative assessment:

  • Calculate signal-to-noise ratios under different conditions

  • Establish reproducible detection thresholds based on controls

• Multiple detection methods:

  • Verify findings with orthogonal techniques (immunofluorescence, mass spectrometry)

  • Use different antibody clones targeting separate epitopes if available

This systematic approach provides confidence in identifying genuine SPCC736.13 signals versus experimental artifacts.

What are potential explanations for contradictory results between ELISA and Western blot when using SPCC736.13 antibody?

Discrepancies between ELISA and Western blot results may arise from multiple factors:

• Epitope accessibility differences:

  • ELISA typically detects native proteins while Western blot detects denatured proteins

  • The epitope recognized by SPCC736.13 antibody may be conformation-dependent

• Sample preparation effects:

  • Protein denaturation during SDS-PAGE may destroy certain epitopes

  • Different buffer compositions between methods may affect antibody binding

• Sensitivity thresholds:

  • ELISA generally offers higher sensitivity than Western blotting

  • Low abundance proteins may be detected by ELISA but not Western blot

• Cross-reactivity profiles:

  • Non-specific binding may differ between the two techniques

  • Different blocking agents and washing conditions affect background differently

When facing contradictory results, researchers should systematically evaluate each of these factors and potentially employ additional validation techniques.

What is the optimal protocol for using SPCC736.13 antibody in immunofluorescence microscopy of fission yeast cells?

While immunofluorescence is not specifically listed among validated applications in the search results, researchers working with fission yeast antibodies typically follow this protocol:

• Cell fixation and permeabilization:

  • Fix cells with 3.7% formaldehyde for 30 minutes at room temperature

  • Wash 3× with PEM buffer (100 mM PIPES, 1 mM EGTA, 1 mM MgSO₄, pH 6.9)

  • Digest cell wall with Zymolyase (1 mg/ml) for 30 minutes at 37°C

  • Permeabilize with 1% Triton X-100 in PEM for 5 minutes

• Antibody incubation:

  • Block with 5% BSA in PEMBAL buffer for 30 minutes

  • Incubate with SPCC736.13 antibody (starting at 1:100 dilution) overnight at 4°C

  • Wash 3× with PEMBAL buffer

  • Incubate with fluorescently-labeled secondary antibody for 2 hours at room temperature

• Imaging considerations:

  • Mount slides with antifade reagent containing DAPI for nuclear counterstaining

  • Include wild-type and SPCC736.13 knockout controls

  • Capture z-stack images to ensure complete cellular visualization

This protocol should be optimized for each experimental system to achieve the best signal-to-noise ratio.

How should researchers prepare S. pombe samples for optimal Western blot detection using SPCC736.13 antibody?

Optimal S. pombe sample preparation for Western blotting includes:

• Cell lysis procedure:

  • Harvest cells in mid-log phase (OD₆₀₀ 0.5-0.8)

  • Wash cells in ice-cold stop buffer (150 mM NaCl, 50 mM NaF, 10 mM EDTA, 1 mM NaN₃)

  • Resuspend in lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% NP-40)

  • Add protease inhibitor cocktail, 1 mM PMSF, and phosphatase inhibitors

  • Disrupt cells with glass beads using a bead beater (5 cycles, 30 seconds on/30 seconds off)

• Sample processing:

  • Clear lysate by centrifugation at 13,000 × g for 15 minutes at 4°C

  • Determine protein concentration using Bradford or BCA assay

  • Normalize all samples to equal protein concentration

• Gel loading preparation:

  • Mix samples with 4× Laemmli buffer containing 10% β-mercaptoethanol

  • Heat at 95°C for 5 minutes

  • Load 20-50 μg total protein per lane

• Transfer and detection optimization:

  • Use PVDF membrane for optimal protein binding

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

  • Incubate with SPCC736.13 antibody overnight at 4°C

  • Wash extensively with TBST (at least 3 × 10 minutes)

  • Detect using appropriate HRP-conjugated secondary antibody and chemiluminescence

This protocol maximizes protein extraction while preserving epitope integrity for optimal antibody detection.

How can SPCC736.13 antibody be used in quantitative proteomics studies of S. pombe?

For quantitative proteomics applications:

• Immunoaffinity enrichment strategy:

  • Couple SPCC736.13 antibody to NHS-activated sepharose or magnetic beads

  • Perform immunoprecipitation from whole cell lysates

  • Elute bound proteins for mass spectrometry analysis

• Sample preparation considerations:

  • Use SILAC or TMT labeling for accurate quantification

  • Include spike-in standards for absolute quantification

  • Prepare biological and technical replicates for statistical validity

• Data analysis approach:

  • Compare SPCC736.13-containing complexes across different conditions

  • Identify significantly enriched interacting partners

  • Validate key interactions with reciprocal immunoprecipitation

• Controls and validation:

  • Include IgG control pulldowns

  • Verify enrichment efficiency by Western blot before mass spectrometry

  • Confirm key findings with orthogonal methods (proximity ligation assay)

This approach enables identification of protein complexes and post-translational modifications associated with SPCC736.13 under different experimental conditions.

What considerations are important when using SPCC736.13 antibody in chromatin immunoprecipitation (ChIP) experiments?

For ChIP applications, researchers should consider:

• Crosslinking optimization:

  • Test different formaldehyde concentrations (0.5-1.5%) and incubation times

  • For protein-protein interactions, consider dual crosslinking with DSG followed by formaldehyde

• Chromatin fragmentation:

  • Optimize sonication conditions to achieve fragments of 200-500 bp

  • Verify fragmentation efficiency by agarose gel electrophoresis

• IP conditions:

  • Pre-clear chromatin with protein A/G beads to reduce background

  • Include input, IgG, and positive control antibody samples

  • Optimize antibody concentration and incubation time

• Washing stringency:

  • Implement increasingly stringent washes to remove non-specific binding

  • Consider including up to 500 mM NaCl in final washes

• Data analysis:

  • Design primers for both positive and negative genomic regions

  • Normalize to input samples

  • Calculate enrichment relative to IgG control

While not specifically validated for ChIP in the search results, these guidelines provide a starting point if researchers wish to explore DNA-binding properties of SPCC736.13 or chromatin-associated complexes.