SPCC126.01c Antibody

What is SPCC126.01c and why is it studied in Schizosaccharomyces pombe?

SPCC126.01c is a protein found in Schizosaccharomyces pombe (fission yeast), specifically in strain 972 / ATCC 24843. This protein is studied in S. pombe as part of fundamental research into eukaryotic cellular processes. S. pombe serves as an excellent model organism for studying eukaryotic cell biology because of its genetic tractability and similarity to human cells in certain key cellular mechanisms.

The SPCC126.01c protein (UniProt accession: O94394) is targeted by researchers investigating various cellular processes in fission yeast . The antibody against this protein enables localization and functional studies that provide insights into basic cellular mechanisms conserved across eukaryotes.

What are the basic specifications of commercially available SPCC126.01c antibodies?

The commercially available SPCC126.01c antibody (product code: CSB-PA530894XA01SXV) has the following specifications:

This antibody is designed for research use only and not for diagnostic or therapeutic procedures .

How should SPCC126.01c antibody be stored to maintain optimal activity?

For optimal maintenance of SPCC126.01c antibody activity, follow these storage guidelines:

• Upon receipt, immediately store the antibody at -20°C or -80°C for long-term storage .

• Avoid repeated freeze-thaw cycles as they can degrade antibody quality and reduce binding efficiency .

• For working aliquots, store at 4°C for up to one month, but prepare small aliquots to minimize freeze-thaw cycles.

• The antibody is supplied in 50% glycerol with PBS (pH 7.4) and 0.03% Proclin 300 as a preservative, which helps maintain stability during storage .

• When removing from freezer storage, allow the antibody to thaw completely at 4°C before use.

Improper storage can lead to decreased sensitivity in applications like Western blotting and ELISA, potentially yielding inconsistent or false-negative results.

What is the optimal Western blotting protocol for SPCC126.01c antibody?

The optimal Western blotting protocol for SPCC126.01c antibody should include the following methodological steps:

• Sample Preparation:

  • Extract total protein from S. pombe cells using a buffer containing protease inhibitors

  • Determine protein concentration using Bradford or BCA assay

  • Prepare 20-50 μg of protein per lane in reducing sample buffer

• Gel Electrophoresis:

  • Use 10-12% SDS-PAGE depending on the molecular weight of SPCC126.01c

  • Run at 100-120V until adequate separation

• Transfer:

  • Transfer proteins to PVDF or nitrocellulose membrane at 100V for 1 hour or 30V overnight at 4°C

  • Verify transfer efficiency with Ponceau S staining

• Blocking:

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

• Primary Antibody Incubation:

  • Dilute SPCC126.01c antibody at 1:500 to 1:2000 in blocking buffer

  • Incubate overnight at 4°C with gentle rocking

• Washing:

  • Wash membrane 3-4 times with TBST, 5-10 minutes each

• Secondary Antibody Incubation:

  • Use anti-rabbit HRP-conjugated secondary antibody at 1:5000 to 1:10000 dilution

  • Incubate for 1 hour at room temperature

• Detection:

  • Develop using enhanced chemiluminescence (ECL) reagent

  • Expose to X-ray film or use an imaging system for detection

This protocol should be optimized based on the specific laboratory conditions and the nature of the samples being analyzed.

How can I troubleshoot weak or absent signal when using SPCC126.01c antibody in Western blots?

When troubleshooting weak or absent signals with SPCC126.01c antibody in Western blots, consider these methodological approaches:

• Protein Expression Level:

  • Confirm that SPCC126.01c is expressed under your experimental conditions

  • Consider enriching the protein using immunoprecipitation before Western blotting

• Antibody Dilution:

  • Test a range of antibody dilutions (e.g., 1:250, 1:500, 1:1000)

  • Reduce the dilution if signal is weak

• Incubation Conditions:

  • Extend primary antibody incubation to 48 hours at 4°C

  • Consider incubating with primary antibody for 2-3 hours at room temperature

• Protein Loading:

  • Increase the amount of total protein loaded (50-100 μg)

  • Verify protein transfer with Ponceau S staining

• Membrane Type:

  • PVDF may provide better sensitivity than nitrocellulose for some applications

  • Low-fluorescence PVDF membranes may improve signal-to-noise ratio for fluorescent detection

• Blocking Reagent:

  • Test alternative blocking agents (BSA, commercial blockers)

  • Reduce blocking time if antibody binding may be affected

• Detection Method:

  • Use high-sensitivity ECL substrates for enhanced detection

  • Consider signal amplification systems for low-abundance proteins

• Antibody Quality:

  • Check antibody expiration date and storage conditions

  • Test a positive control sample where the target protein is known to be expressed

• Sample Preparation:

  • Ensure complete denaturation of samples

  • Include protease inhibitors in lysis buffer to prevent degradation

A systematic approach addressing these factors should help identify the source of the weak signal issue.

How can SPCC126.01c antibody be used for immunofluorescence in S. pombe?

For effective immunofluorescence using SPCC126.01c antibody in S. pombe, follow this methodological approach:

• Cell Fixation and Permeabilization:

  • Fix exponentially growing S. pombe cells with 3.7% formaldehyde for 30 minutes at room temperature

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

  • Permeabilize cell wall with zymolyase (1 mg/ml) in PEMS buffer (PEM + 1.2 M sorbitol) for 30 minutes at 37°C

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

• Blocking:

  • Block with PEMBAL (PEM + 1% BSA, 0.1% sodium azide, 100 mM lysine hydrochloride) for 30 minutes

• Primary Antibody Incubation:

  • Dilute SPCC126.01c antibody 1:100 to 1:500 in PEMBAL

  • Incubate cells overnight at 4°C or 2-3 hours at room temperature

  • Wash 3 times with PEMBAL, 5 minutes each

• Secondary Antibody Incubation:

  • Use fluorophore-conjugated anti-rabbit IgG (e.g., Alexa Fluor 488, 594, or 647) at 1:500 dilution in PEMBAL

  • Incubate for 1-2 hours at room temperature in the dark

  • Wash 3 times with PEMBAL

• Nuclear Staining:

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

  • Wash once with PEM

• Mounting and Imaging:

  • Mount cells in antifade mounting medium

  • Image using confocal or fluorescence microscopy

• Controls:

  • Include secondary antibody-only control to assess background fluorescence

  • Include wild-type and gene deletion strains when possible to verify specificity

This protocol should be optimized based on the specific subcellular localization of SPCC126.01c and the microscopy setup available.

What are the considerations for using SPCC126.01c antibody in chromatin immunoprecipitation (ChIP) experiments?

When using SPCC126.01c antibody for ChIP experiments, consider these methodological factors:

• Crosslinking Optimization:

  • Test different formaldehyde concentrations (0.75% to 1.5%) and incubation times (10-20 minutes)

  • For proteins with indirect DNA interactions, consider using protein-protein crosslinkers like DSG before formaldehyde

• Chromatin Fragmentation:

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

  • Verify fragmentation efficiency by agarose gel electrophoresis

• Antibody Amount and Quality:

  • Test different amounts of SPCC126.01c antibody (2-10 μg per ChIP)

  • Use high-quality, ChIP-validated SPCC126.01c antibody when possible

  • Consider antibody batch variation if reproducibility issues arise

• Preclearing and Controls:

  • Preclear chromatin with protein A/G beads before antibody addition

  • Include IgG control, input sample, and positive control (antibody against known DNA-binding protein)

  • Include a negative control targeting a genomic region not expected to interact with SPCC126.01c

• Washing and Elution:

  • Use stringent washing conditions to reduce background

  • Optimize elution conditions for efficient recovery of target DNA

• Quantification Methods:

  • Design qPCR primers for regions of interest with amplicon sizes of 80-150 bp

  • Consider ChIP-seq for genome-wide analysis of binding sites

• Data Analysis:

  • Calculate enrichment as % input or relative to IgG control

  • For ChIP-seq, use appropriate peak calling algorithms and validate peaks with qPCR

• Specific Considerations for SPCC126.01c:

  • If SPCC126.01c is not a direct DNA-binding protein, consider potential technical challenges

  • If studying protein complexes, sequential ChIP (re-ChIP) may be necessary

The success of ChIP with SPCC126.01c antibody will depend on the protein's biological role and its proximity to chromatin.

How does SPCC126.01c antibody performance compare with antibodies against homologous proteins in other model organisms?

The performance comparison between SPCC126.01c antibody and antibodies against homologous proteins in other model organisms reveals important considerations for cross-species studies:

When planning cross-species studies:

• Epitope Conservation Analysis:

  • Perform sequence alignment of SPCC126.01c with potential homologs

  • Identify conserved regions that might serve as common epitopes

  • Request information about the specific epitope recognized by the antibody

• Validation Requirements:

  • Cross-reactivity must be experimentally validated before use in other species

  • Include positive controls from S. pombe alongside samples from other organisms

  • Consider using tagged recombinant versions of homologous proteins if antibody cross-reactivity is poor

• Application-Specific Considerations:

  • Western blotting may show cross-reactivity more readily than immunofluorescence

  • Denatured epitopes (Western blot) may differ from native epitopes (IP, IF)

  • Higher antibody concentrations may be necessary for non-target species

• Alternative Approaches:

  • For comparative studies, consider using species-specific antibodies for each ortholog

  • Epitope tagging of homologous proteins may provide a more consistent detection method across species

The SPCC126.01c antibody is specifically raised against and validated for S. pombe, making it most reliable for studies in this organism .

How should I analyze conflicting data when using SPCC126.01c antibody across different experimental techniques?

When facing conflicting data from different experimental techniques using SPCC126.01c antibody, implement this methodological framework for resolution:

• Technique-Specific Considerations:

  • Western Blot vs. Immunofluorescence:

    • Different epitope accessibility in native vs. denatured conditions

    • Native protein complexes may mask antibody binding sites

    • Fixation methods in IF may alter epitope recognition

  • Immunoprecipitation vs. Western Blot:

    • Buffer conditions affect antibody-antigen interactions differently

    • Co-immunoprecipitated proteins may interfere with detection

    • IP often requires higher antibody affinity than WB

• Systematic Validation Approach:

  • Confirm antibody specificity using genetic controls (deletion strains)

  • Test multiple commercial SPCC126.01c antibodies targeting different epitopes

  • Validate with orthogonal methods (e.g., epitope tagging, mass spectrometry)

• Data Integration Strategy:

  • Create a confidence matrix ranking results by technique reliability

  • Weight data based on appropriate positive and negative controls

  • Consider biological context when interpreting discrepancies

• Methodological Reconciliation:

  • Modify protocols to standardize conditions across techniques when possible

  • Adjust antibody concentrations based on technique-specific requirements

  • Consider native vs. denaturing conditions as a source of discrepancy

• Advanced Resolution Approaches:

  • Use CRISPR/Cas9 to tag endogenous SPCC126.01c with GFP or other tags

  • Perform functional assays to correlate with antibody-based detection

  • Consider post-translational modifications or isoforms as sources of conflicting results

This systematic approach will help determine whether conflicting data stems from technical limitations or reflects true biological complexity.

How can SPCC126.01c antibody be used to study protein-protein interactions in S. pombe?

To investigate protein-protein interactions involving SPCC126.01c in S. pombe, implement these methodological approaches:

• Co-Immunoprecipitation (Co-IP):

  • Protocol Optimization:

    • Use mild lysis conditions (e.g., 0.5% NP-40 or 1% Triton X-100) to preserve protein complexes

    • Include protease and phosphatase inhibitors to maintain interaction integrity

    • Pre-clear lysates with Protein A/G beads to reduce non-specific binding

    • Incubate lysates with SPCC126.01c antibody (4-10 μg) overnight at 4°C

    • Capture complexes with Protein A/G beads for 2-4 hours

    • Perform stringent washing (4-5 times) to reduce background

    • Elute and analyze interacting proteins by Western blot or mass spectrometry

  • Controls:

    • IgG control to assess non-specific binding

    • Input sample (5-10%) to confirm presence of proteins before IP

    • SPCC126.01c deletion strain as negative control

• Proximity-Dependent Labeling:

  • BioID Approach:

    • Create fusion of SPCC126.01c with BirA* biotin ligase

    • Express in S. pombe and supply biotin to culture medium (50 μM, 16-24 hours)

    • Lyse cells and purify biotinylated proteins using streptavidin beads

    • Identify labeled proteins by mass spectrometry

    • Validate interactions using SPCC126.01c antibody in reverse Co-IP

• Crosslinking Immunoprecipitation:

  • Treat cells with membrane-permeable crosslinkers (DSP, formaldehyde)

  • Lyse cells under denaturing conditions

  • Perform IP with SPCC126.01c antibody

  • Identify crosslinked partners by mass spectrometry

  • Confirm specific interactions by testing candidate interactors

• Two-Hybrid Validation:

  • Use identified interactors in yeast two-hybrid or split-ubiquitin assays

  • Validate direct interactions through in vitro binding assays with recombinant proteins

  • Correlate interaction data with co-localization studies using SPCC126.01c antibody

• Quantitative Analysis:

  • Use SILAC or TMT labeling to quantify differential interactions under various conditions

  • Calculate enrichment ratios compared to controls

  • Focus on reproducible interactions across replicates

This comprehensive approach will provide robust identification of SPCC126.01c interacting partners and insights into its functional role in S. pombe cellular processes.

What considerations are important when using SPCC126.01c antibody to study post-translational modifications?

When investigating post-translational modifications (PTMs) of SPCC126.01c using antibodies, consider these methodological principles:

• PTM-Specific Antibody Limitations:

  • General SPCC126.01c antibody may not distinguish between modified and unmodified forms

  • Consider generating or acquiring PTM-specific antibodies for direct detection

  • Validate PTM-specific antibodies using appropriate controls (e.g., phosphatase-treated samples)

• Enrichment Strategies:

  • Phosphorylation Analysis:

    • Use phosphatase inhibitors during cell lysis (50 mM NaF, 10 mM Na3VO4, 10 mM β-glycerophosphate)

    • Enrich phosphorylated proteins using metal oxide affinity chromatography (MOAC) or phospho-specific antibodies

    • Detect shifts in molecular weight or mobility using Phos-tag™ SDS-PAGE

  • Ubiquitination Detection:

    • Include deubiquitinase inhibitors (NEM, IAA) in lysis buffer

    • Perform IP with SPCC126.01c antibody under denaturing conditions

    • Probe Western blots with anti-ubiquitin antibodies

• Mass Spectrometry Integration:

  • Immunoprecipitate SPCC126.01c using validated antibody

  • Perform in-gel digestion with appropriate proteases (consider multiple proteases for better coverage)

  • Analyze by LC-MS/MS with PTM-specific fragmentation methods (e.g., HCD, ETD)

  • Confirm PTM sites with targeted MS approaches (PRM, MRM)

• Functional Analysis:

  • Correlate PTM detection with specific cellular conditions or stresses

  • Perform site-directed mutagenesis of identified PTM sites

  • Compare wild-type and mutant phenotypes to establish PTM significance

• Technical Challenges and Solutions:

• Temporal and Spatial Considerations:

  • Monitor PTM dynamics during cell cycle or stress responses

  • Compare PTMs across different subcellular fractions

  • Use immunofluorescence with PTM-specific antibodies for spatial information

This systematic approach will enable comprehensive characterization of SPCC126.01c post-translational modifications and their functional significance.

How can SPCC126.01c antibody be used in combination with CRISPR-Cas9 genome editing in S. pombe?

Integrating SPCC126.01c antibody with CRISPR-Cas9 genome editing in S. pombe enables powerful experimental approaches:

• Validation of Genome Editing:

  • Knockout Verification:

    • Use SPCC126.01c antibody to confirm absence of protein in CRISPR knockout strains

    • Compare signal intensity in Western blots between wild-type and knockout strains

    • Include heterozygous strains to assess dose-dependent expression

  • Tag Integration Confirmation:

    • Verify successful integration of epitope tags through dual detection

    • Use SPCC126.01c antibody alongside tag-specific antibodies (e.g., FLAG, HA)

    • Confirm maintained protein expression levels after tag integration

• Functional Domain Analysis:

  • Generate domain deletion mutants using CRISPR-Cas9

  • Use SPCC126.01c antibody to assess expression, stability, and localization

  • Create a panel of truncation mutants to map antibody epitope

• Promoter Modifications:

  • Replace native promoter with regulatable promoters (e.g., nmt1)

  • Use antibody to quantify expression changes under different conditions

  • Correlate expression levels with phenotypic outcomes

• Quantitative Analysis Protocol:

  • Collect samples at defined time points after promoter induction/repression

  • Use standardized Western blot protocol with loading controls

  • Quantify band intensity using digital image analysis software

  • Calculate relative expression normalized to wild-type controls

• Advanced Applications:

  • CRISPRi/CRISPRa:

    • Implement CRISPR interference or activation to modulate SPCC126.01c expression

    • Use antibody to measure expression changes and correlate with phenotype

    • Establish dose-response relationships

  • Base Editing:

    • Introduce specific mutations in SPCC126.01c

    • Use antibody to assess effects on protein stability and interactions

    • Correlate mutations with functional outcomes

• Method Integration Table:

This integrated approach provides robust validation of genome editing while enabling sophisticated functional analysis of SPCC126.01c in S. pombe.