SPCC24B10.20 Antibody

What are the validated applications for SPCC24B10.20 antibody?

SPCC24B10.20 antibody has been validated for Western Blotting (WB) and Enzyme-Linked Immunosorbent Assay (ELISA) applications. These techniques are fundamental for detecting and quantifying the SPCC24B10.20 protein in Schizosaccharomyces pombe samples. When using this antibody for Western blotting, researchers should expect a band at the predicted molecular weight for SPCC24B10.20, though the specific weight is not provided in current literature .

What are the optimal storage conditions for SPCC24B10.20 antibody?

For maximum stability and activity retention, store the SPCC24B10.20 antibody at -20°C or -80°C. Avoid repeated freeze-thaw cycles as these can degrade antibody performance. The antibody is supplied in liquid form with 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . For long-term storage projects, aliquoting the antibody is recommended to minimize freeze-thaw cycles.

What are the recommended positive and negative controls for validating SPCC24B10.20 antibody specificity?

For positive controls, use lysates from wild-type Schizosaccharomyces pombe (strain 972/ATCC 24843) expressing the SPCC24B10.20 protein. For negative controls, utilize SPCC24B10.20 deletion mutants or pre-immune serum. Similar to other antibody validation protocols, comparing signal between wild-type and knockout strains provides definitive evidence of specificity .

How can I optimize Western blot conditions for SPCC24B10.20 antibody in fission yeast samples?

For optimal Western blotting results with SPCC24B10.20 antibody:

• Sample preparation: Extract proteins using a method that preserves native protein structure, such as glass bead lysis in a non-denaturing buffer

• Gel electrophoresis: Use 5-20% SDS-PAGE gradient gels at 70V (stacking)/90V (resolving) for 2-3 hours

• Transfer: Transfer proteins to nitrocellulose membrane at 150mA for 50-90 minutes

• Blocking: Block with 5% non-fat milk in TBS for 1.5 hours at room temperature

• Primary antibody: Incubate with SPCC24B10.20 antibody at 1:500 dilution overnight at 4°C

• Washing: Wash membrane with TBS-0.1% Tween 3 times, 5 minutes each

• Secondary antibody: Incubate with goat anti-rabbit IgG-HRP at 1:5000 dilution for 1.5 hours at room temperature

• Detection: Develop using enhanced chemiluminescence (ECL) detection system

What approaches can be used to determine if SPCC24B10.20 is chromatin-bound in specific cellular contexts?

To investigate chromatin association of SPCC24B10.20:

• Chromatin fractionation: Separate soluble and chromatin-bound proteins through differential centrifugation and detergent extraction

• Chromatin immunoprecipitation (ChIP): Use SPCC24B10.20 antibody to pull down protein-DNA complexes

• Comparative proteomic analysis: Apply techniques similar to those described in Wang et al. (2012) for chromatin-bound protein analysis in S. pombe

• Microscopy validation: Perform immunofluorescence microscopy with SPCC24B10.20 antibody to visualize nuclear localization

This multi-method approach provides complementary evidence for chromatin association and can reveal context-dependent interactions .

How can I troubleshoot cross-reactivity issues with SPCC24B10.20 antibody?

If experiencing cross-reactivity:

• Increase washing stringency with higher salt concentrations (150mM to 300mM NaCl) in wash buffers

• Perform competitive blocking with purified recombinant SPCC24B10.20 protein

• Adjust antibody concentration (try using more diluted antibody)

• Test different blocking reagents (BSA vs. milk vs. commercial blockers)

• Use knockout or depleted samples as definitive negative controls

• Consider peptide competition assays to confirm epitope specificity

Cross-reactivity analysis is critical since antibodies raised against specific epitopes may recognize similar sequences in other proteins .

What is the function of SPCC24B10.20 in Schizosaccharomyces pombe, and why is it relevant for study?

SPCC24B10.20 (UniProt Q9P7I6) is a protein in Schizosaccharomyces pombe with functions relevant to fundamental cellular processes. While detailed functional information is limited in the current literature, its study is valuable because:

• S. pombe is a model organism with conserved molecular mechanisms relevant to human cells

• Understanding SPCC24B10.20 may provide insights into chromatin regulation, as suggested by its potential chromatin association

• Comparative studies between S. pombe proteins and homologs in other species can reveal evolutionarily conserved functions

Research into S. pombe proteins like SPCC24B10.20 contributes to our understanding of fundamental eukaryotic cellular processes .

How does the polyclonal nature of SPCC24B10.20 antibody impact experimental design and data interpretation?

The polyclonal nature of SPCC24B10.20 antibody has several implications:

• Multiple epitope recognition: Polyclonal antibodies recognize multiple epitopes on the target protein, potentially increasing sensitivity but also raising specificity concerns

• Batch variability: Different lots may have varying affinities and epitope specificities

• Cross-reactivity potential: Higher chance of recognizing similar epitopes on non-target proteins

• Detection robustness: Less affected by epitope masking due to protein conformational changes or post-translational modifications

When designing experiments:

• Include proper controls for each new antibody lot

• Consider epitope availability in different experimental conditions

• Be aware that polyclonal responses may detect modified forms of the protein differentially

How can I quantitatively analyze SPCC24B10.20 protein levels in different experimental conditions?

For quantitative analysis of SPCC24B10.20:

• Western blot quantification:

  • Use internal loading controls (e.g., tubulin, actin)

  • Apply densiometric analysis with appropriate software (ImageJ, Image Lab)

  • Ensure linear detection range through pilot experiments

  • Normalize to total protein using stain-free technology or Ponceau staining

• ELISA-based quantification:

  • Develop a standard curve using recombinant SPCC24B10.20

  • Ensure sample dilutions fall within the linear range of detection

  • Account for matrix effects through spike-and-recovery experiments

• Statistical analysis:

  • Apply appropriate statistical tests based on experimental design

  • Account for biological and technical replicates

  • Consider non-parametric tests for small sample sizes

What approaches can be used to study protein-protein interactions involving SPCC24B10.20?

To investigate protein-protein interactions of SPCC24B10.20:

• Co-immunoprecipitation (Co-IP):

  • Use SPCC24B10.20 antibody to pull down the protein and its binding partners

  • Analyze by mass spectrometry or Western blot with antibodies against suspected interactors

• Proximity-based labeling:

  • Create fusion proteins with BioID or APEX2

  • Identify proteins in close proximity through biotinylation and streptavidin pulldown

• Yeast two-hybrid screening:

  • Use SPCC24B10.20 as bait to screen for interacting proteins

• Fluorescence resonance energy transfer (FRET):

  • Tag SPCC24B10.20 and potential partners with appropriate fluorophores

  • Measure energy transfer as indicator of protein proximity

These complementary approaches provide a comprehensive view of the protein's interaction network .

How can SPCC24B10.20 antibody be used in ChIP-seq experiments to map genomic binding sites?

To perform ChIP-seq with SPCC24B10.20 antibody:

• Experimental design:

  • Cross-link proteins to DNA in vivo using formaldehyde

  • Sonicate chromatin to 200-500bp fragments

  • Immunoprecipitate with SPCC24B10.20 antibody

  • Reverse cross-links and purify DNA

  • Prepare sequencing libraries and perform deep sequencing

• Controls and validation:

  • Include input DNA control

  • Use IgG or pre-immune serum as negative control

  • Validate enrichment at selected loci by ChIP-qPCR before sequencing

  • Consider spike-in normalization for quantitative comparisons

• Data analysis:

  • Align reads to S. pombe genome

  • Call peaks using appropriate algorithms (MACS2, HOMER)

  • Perform motif analysis to identify potential DNA binding motifs

  • Correlate binding sites with gene expression data

This approach can determine if SPCC24B10.20 has specific genomic targets, potentially revealing its function in gene regulation .

How can I compare results between different antibodies targeting SPCC24B10.20?

When comparing results between different SPCC24B10.20 antibodies:

• Epitope mapping:

  • Determine which regions of the protein each antibody recognizes

  • Consider how epitope location might affect detection in different applications

• Validation standards:

  • Use identical positive and negative controls for each antibody

  • Test under standardized conditions to enable direct comparisons

• Performance metrics to compare:

  • Sensitivity (limit of detection)

  • Specificity (cross-reactivity profile)

  • Signal-to-noise ratio

  • Reproducibility across experiments

• Documentation:

  • Create a comparative table with standardized metrics

  • Note batch numbers and validation dates

This systematic approach enables objective assessment of antibody performance for specific applications .

What methodological considerations are important when studying potential orthologs of SPCC24B10.20 in other species?

When investigating potential orthologs:

• Sequence analysis:

  • Perform sequence alignment and phylogenetic analysis

  • Identify conserved domains and motifs

  • Consider structural predictions for functional conservation

• Antibody cross-reactivity assessment:

  • Test SPCC24B10.20 antibody against recombinant ortholog proteins

  • Validate with species-specific positive and negative controls

• Functional comparison:

  • Design comparative experiments to test conserved functions

  • Use complementation studies in knockout/knockdown models

• Expression pattern analysis:

  • Compare tissue/cellular localization between species

  • Analyze expression under similar experimental conditions

This approach helps establish functional conservation and divergence, providing evolutionary context for SPCC24B10.20's role .

How can advanced proteomics approaches be integrated with SPCC24B10.20 antibody-based studies?

Integration of proteomics with antibody-based approaches:

• Antibody-based enrichment for targeted proteomics:

  • Use SPCC24B10.20 antibody for immunoprecipitation

  • Analyze by mass spectrometry for interacting partners

  • Identify post-translational modifications

• Validation of proteomics findings:

  • Confirm mass spectrometry-identified interactions by Co-IP/Western blot

  • Validate expression changes through orthogonal methods

• Integrated experimental design:

  • Map modification sites identified by proteomics

  • Generate modification-specific antibodies for functional studies

  • Correlate protein interaction networks with functional assays

• Data integration framework:

  Technique	Primary Data	Validation Method	Functional Follow-up
  IP-MS	Interaction partners	Co-IP/Western blot	Functional assays
  PTM-MS	Modification sites	Site-specific antibodies	Mutagenesis studies
  ChIP-seq	Genomic binding sites	ChIP-qPCR	Reporter assays
  Proteomics	Expression changes	Western blot	Phenotypic analysis