SPBCPT2R1.04c Antibody

What is SPBCPT2R1.04c and its biological significance in S. pombe?

SPBCPT2R1.04c is a protein found in Schizosaccharomyces pombe (fission yeast), identified by the Uniprot accession number P0CT99. While complete characterization data is limited in public databases, it belongs to the wider class of proteins studied in S. pombe, which serves as an important model organism for eukaryotic cellular processes. The protein is part of the extensive catalog of S. pombe proteins that are used to study fundamental cellular mechanisms, similar to other S. pombe proteins like those coded by SPAC1F5.11c, SPAC1F7.10, and SPAC12G12.16c .

How is SPBCPT2R1.04c Antibody validated for research applications?

Antibody validation typically involves multiple complementary approaches:

• Western blot analysis: Demonstrating specific binding to the target protein at the expected molecular weight with minimal cross-reactivity

• Knockout/knockdown controls: Testing on samples where the target protein is absent or reduced

• Immunoprecipitation followed by mass spectrometry: Confirming capture of the intended target

• Peptide competition assays: Verifying specificity by blocking antibody binding with the immunizing peptide

For S. pombe antibodies like SPBCPT2R1.04c, validation is particularly important as they are used in fundamental research contexts where false positives can lead to significant misinterpretations. Validation should include controls similar to those used in other antibody research, as indicated by methodologies described for antibody characterization in databases like PLAbDab .

What experimental controls are essential when using SPBCPT2R1.04c Antibody?

When designing experiments with SPBCPT2R1.04c Antibody, researchers should incorporate:

Positive controls:

• Known positive samples of S. pombe expressing the target protein

• Recombinant SPBCPT2R1.04c protein (if available)

Negative controls:

• Isotype control antibodies to detect non-specific binding

• Wild-type vs. knockout/knockdown S. pombe strains

• Primary antibody omission controls

Additional controls:

• Serial dilution series to establish detection limits

• Pre-absorption controls with immunizing peptide

• Cross-species samples to evaluate specificity

Proper controls are particularly crucial for antibody-based detection methods in S. pombe research, where cross-reactivity with other yeast proteins can confound results .

How do epitope accessibility issues affect SPBCPT2R1.04c Antibody binding efficiency in different experimental conditions?

Epitope accessibility can significantly impact antibody binding efficiency, particularly in techniques involving different sample preparation methods:

Factors affecting epitope accessibility:

• Protein folding and tertiary structure

• Post-translational modifications

• Protein-protein interactions

• Fixation and sample preparation methods

For SPBCPT2R1.04c Antibody, optimizing epitope accessibility might require:

• For fixed samples (IF/IHC): Testing multiple fixation approaches (paraformaldehyde, methanol, or acetone) at different concentrations and durations

• For denatured samples (Western blot): Comparing reducing vs. non-reducing conditions

• For native applications (IP): Using mild detergents that preserve protein conformation while allowing antibody access

Researchers should perform systematic optimization similar to approaches used with other S. pombe antibodies and document conditions that maximize signal-to-noise ratio while maintaining specificity .

What are the methodological considerations for using SPBCPT2R1.04c Antibody in protein-protein interaction studies?

When using SPBCPT2R1.04c Antibody for protein-protein interaction studies, researchers should consider:

Experimental approaches:

• Co-immunoprecipitation (Co-IP):

  • Use mild lysis buffers to preserve interactions

  • Consider crosslinking for transient interactions

  • Include RNase/DNase treatment to eliminate nucleic acid-mediated associations

• Proximity ligation assay (PLA):

  • Requires complementary antibodies against interaction partners

  • Offers high sensitivity for detecting protein interactions in situ

• Pull-down assays:

  • May require optimization of salt and detergent concentrations

  • Tag-based approaches can complement antibody-based methods

Data interpretation:

• Include appropriate negative controls to distinguish specific from non-specific interactions

• Consider reciprocal IP experiments to confirm interactions

• Validate interactions using orthogonal methods

These approaches are consistent with methodologies used in antibody-based interaction studies across model organisms, including yeast systems like S. pombe .

How can researchers address batch-to-batch variability of SPBCPT2R1.04c Antibody?

Batch-to-batch variability is a significant challenge in antibody-based research. For SPBCPT2R1.04c Antibody, researchers should:

• Establish standardized validation protocols:

  • Create a detailed validation checklist for each new batch

  • Document minimum performance criteria for key applications

• Maintain reference samples:

  • Preserve positive control lysates from successful experiments

  • Create standard curves with established batches

• Implement quality control measures:

  • Compare new batches against previous ones using parallel experiments

  • Document lot numbers and create internal reference standards

• Statistical approaches:

  • Use statistical methods to normalize data across batches

  • Include batch information in experimental design and analysis

Addressing variability is critical for longitudinal studies and for comparing results across publications, particularly in specialized antibodies like those targeting S. pombe proteins .

What is the optimized Western blot protocol for SPBCPT2R1.04c Antibody?

Optimized Western Blot Protocol for SPBCPT2R1.04c Antibody:

Sample preparation:

• Harvest S. pombe cells during appropriate growth phase

• Lyse cells in buffer containing:

  • 50 mM HEPES pH 7.5

  • 150 mM NaCl

  • 1 mM EDTA

  • 1% Triton X-100

  • Protease inhibitor cocktail

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

Gel electrophoresis and transfer:

• Separate proteins on 10-12% SDS-PAGE

• Transfer to PVDF membrane (0.45 μm) at 100V for 60 minutes

Immunodetection:

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

• Incubate with SPBCPT2R1.04c Antibody (1:1000 dilution) overnight at 4°C

• Wash 3× with TBST, 10 minutes each

• Incubate with HRP-conjugated secondary antibody for 1 hour at room temperature

• Wash 3× with TBST, 10 minutes each

• Develop using ECL substrate

Optimization considerations:

• Test different antibody dilutions (1:500 to 1:5000)

• Compare blocking agents (milk vs. BSA)

• Evaluate various incubation times (1 hour to overnight)

This protocol incorporates general best practices for antibody-based detection and should be optimized for the specific research context .

How can SPBCPT2R1.04c Antibody be effectively used in immunofluorescence studies with S. pombe?

Immunofluorescence Protocol for S. pombe using SPBCPT2R1.04c Antibody:

Cell preparation:

• Grow S. pombe to mid-log phase (OD600 = 0.5-0.7)

• 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 for 5 minutes

Immunostaining:

• Block with 5% BSA in PBS for 1 hour

• Incubate with SPBCPT2R1.04c Antibody (1:100 dilution) overnight at 4°C

• Wash 3× with PBS-T

• Incubate with fluorophore-conjugated secondary antibody for 1 hour

• Counterstain with DAPI (1 μg/ml) for 5 minutes

• Mount using antifade mounting medium

Optimization strategies:

• Compare different fixation methods (formaldehyde vs. methanol)

• Test various cell wall digestion conditions

• Evaluate antibody concentrations and incubation times

• Include proper controls (no primary antibody, isotype control)

This protocol is designed based on standard procedures for S. pombe immunofluorescence and should be adjusted based on experimental results .

What troubleshooting approaches address common issues with SPBCPT2R1.04c Antibody in experimental applications?

Common Issues and Troubleshooting Strategies:

Researchers should implement systematic troubleshooting by changing one variable at a time and documenting conditions that resolve the issue, similar to approaches used with other S. pombe antibodies .

How can SPBCPT2R1.04c Antibody be incorporated into ChIP-Seq studies for chromatin-associated proteins?

Chromatin immunoprecipitation sequencing (ChIP-Seq) using SPBCPT2R1.04c Antibody requires careful optimization, particularly if the target protein interacts with chromatin:

ChIP-Seq workflow optimization:

• Crosslinking optimization:

  • Test formaldehyde concentrations (0.5-2%)

  • Evaluate crosslinking times (5-20 minutes)

• Chromatin fragmentation:

  • Compare sonication vs. enzymatic digestion

  • Aim for fragments of 200-500 bp

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads

  • Use 2-5 μg antibody per IP

  • Include IgG control and input samples

• Validation:

  • Perform qPCR validation of enriched regions before sequencing

  • Include biological replicates for statistical power

This application requires high antibody specificity to avoid false positives in genome-wide binding profiles. Verification of antibody specificity using the methods described in PLAbDab would be essential before proceeding with ChIP-Seq experiments .

What are the considerations for using SPBCPT2R1.04c Antibody in quantitative proteomics studies?

When incorporating SPBCPT2R1.04c Antibody into quantitative proteomics workflows:

Methodological considerations:

• Immunoprecipitation-Mass Spectrometry (IP-MS):

  • Use crosslinking approaches to stabilize antibody-bead interactions

  • Include appropriate negative controls (IgG, knockout samples)

  • Consider SILAC or TMT labeling for quantitative comparison

• Antibody-based enrichment prior to LC-MS/MS:

  • Optimize elution conditions to maximize recovery

  • Evaluate non-specific binding to beads or antibody

  • Consider on-bead digestion to minimize sample loss

• Data analysis:

  • Implement stringent filtering criteria for interactors

  • Compare enrichment ratios against appropriate controls

  • Validate key interactions using orthogonal methods

These approaches allow researchers to identify not only the target protein but also its interacting partners, providing insights into protein complexes and networks in S. pombe .

How can SPBCPT2R1.04c Antibody be utilized for studying protein dynamics during cell cycle progression in S. pombe?

S. pombe is a powerful model for cell cycle studies, and SPBCPT2R1.04c Antibody can be employed to track protein dynamics:

Experimental approaches:

• Synchronization methods:

  • Nitrogen starvation and release

  • Hydroxyurea block and release

  • cdc25-22 temperature-sensitive mutant

  • Size selection by centrifugal elutriation

• Analysis techniques:

  • Time-course Western blotting for protein level changes

  • Immunofluorescence to track subcellular localization

  • Live-cell imaging with complementary fluorescent markers

  • Quantitative mass spectrometry for post-translational modifications

• Data quantification:

  • Densitometry analysis of Western blots

  • Automated image analysis for cellular distribution

  • Statistical methods for time-course data

This application requires careful validation of the antibody's specificity across different cell cycle stages and conditions to ensure that observed changes reflect true biological dynamics rather than technical artifacts .

How does research using SPBCPT2R1.04c Antibody complement other approaches to studying S. pombe biology?

SPBCPT2R1.04c Antibody research can be integrated with other S. pombe research approaches to create a comprehensive understanding:

Complementary methodologies:

• Genetic approaches:

  • CRISPR/Cas9 gene editing to create knockouts/knock-ins

  • Conditional mutants (temperature-sensitive, auxin-inducible)

  • Suppressor screening to identify genetic interactions

• Biochemical methods:

  • In vitro reconstitution of protein complexes

  • Structural studies (X-ray crystallography, Cryo-EM)

  • Enzymatic assays for functional characterization

• Systems biology:

  • Transcriptomics to correlate protein and mRNA levels

  • Metabolomics to link protein function to cellular metabolism

  • Mathematical modeling of relevant pathways

What databases and resources can complement research findings using SPBCPT2R1.04c Antibody?

Researchers working with SPBCPT2R1.04c Antibody should leverage these complementary resources:

Key databases and resources:

• PomBase:

  • Comprehensive S. pombe genome database

  • Functional annotations and phenotype data

  • Literature curation and community input

• PLAbDab (Patent and Literature Antibody Database):

  • Repository of functionally diverse antibody sequences

  • Literature-annotated antibody information

  • Growing resource with ~150,000 entries

• Proteomics resources:

  • PeptideAtlas for S. pombe proteomics data

  • PRIDE repository for data deposition

  • STRING database for protein interaction networks

• Microscopy resources:

  • OME Bio-Formats for image data standardization

  • ImageJ/Fiji for quantitative image analysis

  • S. pombe image repositories and phenotype databases

These resources provide context for antibody-based findings and help integrate results into the broader understanding of S. pombe biology .