SPAC977.05c Antibody

What is SPAC977.05c and why is it significant in S. pombe research?

SPAC977.05c is a gene in Schizosaccharomyces pombe (strain 972/ATCC 24843) that encodes a VEL1-related protein. This protein is significant in fission yeast studies as part of understanding fundamental cellular processes in this model organism. The protein has been identified through genomic analysis, though its complete functional characterization is still evolving in the research community . Studying this protein contributes to our understanding of conserved eukaryotic cellular mechanisms, as S. pombe is a valuable model organism with many conserved pathways relevant to human cell biology.

What are the validated applications for SPAC977.05c antibody?

The SPAC977.05c antibody has been validated for several research applications:

These applications have been validated using recombinant SPAC977.05c protein from Schizosaccharomyces pombe (strain 972/ATCC 24843) . The antibody is particularly useful for ensuring identification of the antigen in complex biological samples.

What controls should be included when using SPAC977.05c antibody?

When using the SPAC977.05c antibody, several controls are essential for experimental validity:

• Positive control: Lysate from wild-type S. pombe expressing the native protein.

• Negative control: Lysate from SPAC977.05c deletion strain if available.

• Loading control: An antibody against a constitutively expressed S. pombe protein (e.g., tubulin or actin).

• Secondary antibody control: Primary antibody omission to verify secondary antibody specificity.

• Blocking peptide control: Pre-incubation of the antibody with the immunizing peptide to demonstrate specificity.

These controls ensure that any signals detected are specific to the SPAC977.05c protein and not due to non-specific binding or technical artifacts.

How should SPAC977.05c antibody be stored and handled?

The SPAC977.05c antibody requires specific storage and handling conditions to maintain its performance:

• Storage temperature: Upon receipt, store at -20°C or -80°C.

• Avoid repeated freeze-thaw cycles: Aliquot the antibody upon first thaw to minimize damage.

• Storage buffer: The antibody is provided in a buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative .

• Working dilution preparation: When preparing working dilutions, use a buffer containing a carrier protein like BSA (0.1-1%).

• Shelf life: When properly stored, the antibody retains activity for approximately 12 months.

Proper storage and handling are critical for maintaining antibody performance, particularly for polyclonal antibodies like the SPAC977.05c antibody.

What is the optimal dilution range for SPAC977.05c antibody in Western blot applications?

While specific dilution recommendations may vary slightly between lots, the following dilution ranges serve as starting points for optimization:

Optimization should include a titration experiment where several dilutions are tested simultaneously against the same sample. The optimal dilution provides the strongest specific signal with minimal background staining. For Western blot applications, including a gradient of sample concentrations can help determine the linear detection range of the antibody at different dilutions.

How can cross-reactivity with other S. pombe proteins be assessed and mitigated when using SPAC977.05c antibody?

Cross-reactivity assessment and mitigation requires a systematic approach:

Assessment methodology:

• Immunoblotting with recombinant proteins: Test the antibody against purified recombinant SPAC977.05c and closely related S. pombe proteins.

• Comparative analysis with knockout strains: Compare immunoblot patterns between wild-type and SPAC977.05c deletion strains.

• Mass spectrometry validation: Perform immunoprecipitation followed by mass spectrometry to identify all proteins captured by the antibody.

Mitigation strategies:

• Epitope mapping: Identify the specific epitope(s) recognized by the antibody to predict potential cross-reactivity based on sequence similarity.

• Pre-absorption: Pre-incubate the antibody with recombinant proteins containing cross-reactive epitopes before use.

• Optimization of stringency conditions: Adjust washing buffers and blocking conditions to reduce non-specific binding.

• Alternative detection methods: Validate findings using complementary approaches like mass spectrometry or tagged protein expression.

These approaches help ensure that experimental observations truly reflect the behavior of SPAC977.05c rather than cross-reactive proteins.

What are the optimal sample preparation techniques for detecting SPAC977.05c in S. pombe lysates?

The effective detection of SPAC977.05c requires careful sample preparation, particularly given the unique cell wall characteristics of fission yeast:

Cell lysis protocol:

• Mechanical disruption: Glass bead beating (0.5mm beads) for 5-6 cycles of 30 seconds with 30-second cooling intervals.

• Lysis buffer composition: 50mM HEPES pH 7.5, 150mM NaCl, 1mM EDTA, 1% Triton X-100, 10% glycerol, supplemented with:

  • Protease inhibitor cocktail (complete, EDTA-free)

  • Phosphatase inhibitors (2mM Na₃VO₄, 50mM NaF)

  • 1mM PMSF (added fresh)

• Incubation conditions: Keep samples on ice throughout the preparation process.

• Clarification: Centrifuge at 14,000g for 15 minutes at 4°C to remove cell debris.

Protein quantification and preparation for electrophoresis:

• Quantification method: BCA or Bradford assay, with BSA as standard.

• Sample denaturing: Mix with Laemmli sample buffer (final concentration: 62.5mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.01% bromophenol blue) and heat at 95°C for 5 minutes.

• Loading amount: 20-30μg of total protein per lane for standard detection.

This protocol maximizes protein extraction while minimizing degradation and post-extraction modifications that could affect antibody recognition.

How can epitope masking be addressed when SPAC977.05c protein interactions or modifications interfere with antibody detection?

Epitope masking can significantly impact SPAC977.05c detection, particularly if the protein participates in protein complexes or undergoes post-translational modifications:

Identification of epitope masking:

• Comparative analysis: Compare detection under native versus denaturing conditions.

• Fractionation experiments: Analyze different cellular fractions for differential detection efficiency.

• Co-immunoprecipitation followed by Western blot: Identify potential interacting partners that may mask epitopes.

Resolution strategies:

• Sample preparation modifications:

  • Increase SDS concentration (up to 2%) in sample buffer

  • Extend heating time (up to 10 minutes at 95°C)

  • Add protein denaturants like 8M urea or 6M guanidine HCl

• Epitope retrieval techniques:

  • Mild enzymatic treatment (e.g., lambda phosphatase for phospho-epitopes)

  • pH shifts using acidic or basic buffers

  • Use of detergents with different properties (CHAPS, deoxycholate)

• Alternative antibody approaches:

  • Use multiple antibodies targeting different epitopes

  • Consider using antibodies against tags if working with tagged versions of the protein

These approaches can help overcome epitope masking issues that might otherwise lead to false negative results or underestimation of protein abundance.

What considerations are important when designing co-localization studies using SPAC977.05c antibody in S. pombe?

Co-localization studies with SPAC977.05c antibody require careful planning and execution:

Technical considerations:

• Fixation optimization: Compare different fixation methods:

  • 4% paraformaldehyde (10-15 minutes)

  • Methanol (-20°C for 6 minutes)

  • Combined formaldehyde-methanol approaches

• Permeabilization protocol: Test different approaches:

  • 0.1% Triton X-100 (5-10 minutes)

  • 0.5% Saponin (10 minutes)

  • Enzymatic cell wall digestion with zymolyase (1mg/ml for 10-30 minutes)

• Blocking optimization: Use 5% BSA or 5-10% normal serum from the species of the secondary antibody host.

• Signal amplification options:

  • Tyramide signal amplification

  • Use of high-sensitivity detection systems

Experimental design considerations:

• Selection of co-localization markers: Choose markers for relevant subcellular compartments based on predicted function.

• Controls for spatial resolution: Include samples with known non-overlapping and partially overlapping distributions.

• Quantitative co-localization analysis: Use software like ImageJ with Coloc2 or JACoP plugins for quantitative assessment.

• Super-resolution approaches: Consider STORM, PALM, or SIM for precise co-localization assessment.

These considerations help ensure that co-localization studies provide reliable and interpretable data about SPAC977.05c subcellular distribution and interaction partners.

How can the SPAC977.05c antibody be validated for chromatin immunoprecipitation (ChIP) applications?

Validating the SPAC977.05c antibody for ChIP applications requires a systematic approach, especially if the protein might have DNA-binding properties or chromatin association:

Preliminary validation steps:

• Antibody specificity confirmation:

  • Western blot against nuclear extracts

  • Immunoprecipitation followed by mass spectrometry

  • Comparison with tagged protein control

• ChIP protocol optimization:

  Parameter	Options to Test
  Crosslinking	1% formaldehyde for 5, 10, or 15 minutes
  Sonication	Optimization of cycles and amplitude for 200-500bp fragments
  Antibody amount	2-10μg per reaction
  Incubation time	2 hours vs. overnight
  Wash stringency	Low, medium, and high salt washes

• Controls to include:

  • Input DNA (pre-immunoprecipitation)

  • IgG negative control

  • ChIP with tagged version of SPAC977.05c

  • ChIP in SPAC977.05c deletion strain

Validation experiments:

These validation steps ensure that any ChIP data obtained with the SPAC977.05c antibody accurately reflects the genomic associations of the protein.

What are common causes of high background when using SPAC977.05c antibody in immunoblotting?

High background is a common challenge when working with polyclonal antibodies like the SPAC977.05c antibody. Systematic troubleshooting includes:

Common causes and solutions:

• Insufficient blocking:

  • Increase blocking time from 1 hour to overnight

  • Test alternative blocking agents (5% milk, 5% BSA, commercial blockers)

  • Add 0.1-0.5% Tween-20 to blocking buffer

• Non-specific antibody binding:

  • Increase antibody dilution (test 2-5x more dilute)

  • Pre-absorb with S. pombe lysate from knockout strain

  • Add 0.1-0.2% Tween-20 to antibody dilution buffer

• Inadequate washing:

  • Increase wash duration and number (5-6 washes of 10 minutes each)

  • Use higher detergent concentration in wash buffer (up to 0.1% SDS or 0.3% Tween-20)

  • Consider using TBS instead of PBS if phosphoproteins are studied

• Detection system issues:

  • Reduce exposure time

  • Decrease secondary antibody concentration

  • Test alternative detection systems (chemiluminescence vs. fluorescence)

• Sample preparation problems:

  • Ensure complete protein denaturation

  • Filter lysates to remove particulates

  • Include additional protease inhibitors to prevent degradation products

Methodical testing of these variables helps identify the specific factors contributing to high background in each experimental system.

How can signal variability between experiments be reduced when using SPAC977.05c antibody?

Consistent signal detection is crucial for quantitative analyses. Minimizing variability requires:

Standardization approaches:

• Antibody handling:

  • Prepare single-use aliquots to avoid freeze-thaw cycles

  • Standardize antibody dilution procedures

  • Use consistent antibody incubation times and temperatures

• Sample preparation standardization:

  • Harvest cells at consistent density and growth phase

  • Use standardized lysis protocols with timed steps

  • Process all samples simultaneously when possible

• Experimental controls:

  • Include standard curve of recombinant SPAC977.05c protein

  • Use consistent positive control in every experiment

  • Normalize to multiple loading controls

• Technical standardization:

  Parameter	Standardization Approach
  Gel loading	Use fixed protein amount (25-30μg)
  Transfer conditions	Use same transfer time and current
  Blocking	Standardize blocking time and temperature
  Antibody incubation	Use fixed volumes and container types
  Detection	Use consistent exposure method and times

• Data normalization strategies:

  • Normalize to total protein (Ponceau S or Stain-Free technology)

  • Use multiple housekeeping proteins for normalization

  • Consider normalization to recombinant protein standard curve

Implementing these standardization approaches significantly reduces inter-experimental variability and improves quantitative reliability.

How should results be interpreted when contradictory data is obtained between SPAC977.05c antibody detection and other methods?

Contradictory results between antibody-based detection and other methods require careful investigation:

Systematic analysis approach:

This approach helps researchers interpret contradictory results in a scientifically rigorous manner rather than simply discarding inconvenient data.

How can SPAC977.05c antibody be adapted for proximity labeling studies in fission yeast?

Proximity labeling offers insights into protein interaction networks and requires specific adaptations for use with the SPAC977.05c antibody:

Implementation strategies:

• Antibody-enzyme conjugation approaches:

  • Direct conjugation to HRP for APEX2-based proximity labeling

  • Conjugation to biotin ligase (BioID, TurboID) for biotin-based proximity labeling

  • Use of secondary antibodies conjugated to labeling enzymes

• Optimization parameters:

  Parameter	Considerations
  Conjugation chemistry	NHS ester, maleimide, or click chemistry approaches
  Enzyme:antibody ratio	Test different ratios (3:1, 5:1, 10:1)
  Labeling substrate	Biotin-phenol concentration and incubation time
  Quenching	Rapid termination with quenchers and antioxidants

• Cell permeabilization options:

  • Digitonin (25-50μg/ml) for selective plasma membrane permeabilization

  • Mild detergents like saponin (0.1%)

  • Streptolysin O for reversible pore formation

• Controls and validation:

  • Unconjugated antibody controls

  • Non-specific IgG-enzyme conjugates

  • Comparison with known interaction partners

  • Validation by alternative interaction detection methods

These adaptations allow researchers to map the protein interaction neighborhood of SPAC977.05c with spatial and temporal resolution that complements traditional co-immunoprecipitation approaches.

What strategies can be employed to use SPAC977.05c antibody in studying protein dynamics during cell cycle progression?

Studying protein dynamics during the cell cycle requires specialized approaches:

Experimental design considerations:

• Cell synchronization methods for S. pombe:

  • Lactose gradient centrifugation (size-based selection)

  • Nitrogen starvation followed by release

  • Hydroxyurea block and release

  • cdc25 temperature-sensitive mutant synchronization

• Sampling strategy:

  • Collect samples at 10-20 minute intervals over 3-4 hours

  • Monitor synchrony using microscopic examination and septation index

  • Parallel samples for protein analysis and cell cycle markers

• Multi-parameter analysis:

  • Combine antibody detection with DNA content analysis

  • Include antibodies against cell cycle markers (Cdc13, Cdc2)

  • Monitor post-translational modifications with phospho-specific antibodies

• Quantitative assessment approaches:

  • Normalize SPAC977.05c levels to stable reference proteins

  • Plot relative protein abundance against cell cycle progression

  • Compare with transcript levels if available

• Advanced imaging approaches:

  • Time-lapse immunofluorescence microscopy with cell cycle markers

  • FRAP (Fluorescence Recovery After Photobleaching) if using fluorescently tagged versions

  • Single-cell immunofluorescence quantification with cell cycle staging

These approaches allow researchers to determine whether SPAC977.05c undergoes cell cycle-dependent changes in abundance, localization, or modification state.

How can SPAC977.05c antibody be utilized in multiplexed protein detection systems?

Multiplexed detection systems allow simultaneous analysis of multiple proteins and require specific considerations:

Implementation approaches:

• Fluorescence-based multiplexing:

  • Directly conjugate SPAC977.05c antibody with distinct fluorophores

  • Use secondary antibodies from different species with non-overlapping fluorophores

  • Consider zenon labeling technology for antibodies from the same species

• Mass cytometry adaptation:

  • Conjugate SPAC977.05c antibody with distinct metal isotopes

  • Validate metal-conjugated antibody specificity

  • Develop compensation panels for signal spillover

• Sequential multiplexing strategies:

  • Strip and reprobe membranes (optimize stripping conditions to preserve epitopes)

  • Use tyramide signal amplification with sequential detection

  • Develop spectral unmixing protocols for overlapping signals

• Imaging mass spectrometry integration:

  • Label with isotope-tagged antibodies for MIBI-TOF analysis

  • Develop protocols for tissue or cell preparation compatible with both antibody binding and mass spectrometry

These multiplexed approaches enable researchers to simultaneously analyze SPAC977.05c alongside other proteins of interest, providing insights into complex regulatory networks and protein interactions.

What considerations are important for developing quantitative ELISA assays using SPAC977.05c antibody?

Developing a quantitative ELISA for SPAC977.05c requires careful optimization:

Assay development parameters:

• Antibody pair selection:

  • Test SPAC977.05c antibody as capture or detection antibody

  • Consider using tagged recombinant SPAC977.05c for validated antibody pairs

  • Evaluate monoclonal alternatives if available for better specificity

• Protocol optimization:

  Parameter	Optimization Range
  Coating concentration	1-10μg/ml
  Blocking buffer	Test BSA, casein, and commercial blockers
  Sample dilution	Serial dilutions to determine linear range
  Antibody concentration	Titrate for optimal signal:noise ratio
  Incubation conditions	Time (1-16h) and temperature (4°C-RT)

• Standard curve development:

  • Use purified recombinant SPAC977.05c protein

  • Prepare standard curve in matrix similar to samples

  • Determine lower limit of detection and quantification

  • Assess recovery of spiked standards in sample matrix

• Validation parameters:

  • Intra-assay precision (CV <10%)

  • Inter-assay precision (CV <15%)

  • Specificity (cross-reactivity assessment)

  • Linearity of dilution

  • Spike recovery (80-120%)

• Sample preparation considerations:

  • Optimal lysis buffer composition

  • Potential need for sample pre-clearing

  • Assessment of interfering substances

  • Stability of analyte during storage

These optimization steps ensure development of a robust quantitative ELISA that can reliably measure SPAC977.05c protein levels across various experimental conditions.

How might new antibody engineering technologies be applied to improve SPAC977.05c detection specificity?

Emerging antibody technologies offer potential improvements for SPAC977.05c detection:

Advanced antibody approaches:

• Single-domain antibody development:

  • Nanobodies derived from camelid antibodies

  • Single-chain variable fragments (scFvs)

  • Designed ankyrin repeat proteins (DARPins)

• Affinity maturation strategies:

  • Phage display selection with stringent conditions

  • Yeast surface display with negative selection against similar proteins

  • Directed evolution approaches to optimize binding domains

• Epitope-focused engineering:

  • Design antibodies targeting unique regions of SPAC977.05c

  • Structure-guided antibody design

  • Computational epitope prediction and antibody design

• Multispecific antibody formats:

  • Bispecific antibodies targeting SPAC977.05c and interaction partners

  • Antibody-oligonucleotide conjugates for proximity detection

  • Split-epitope recognition systems

These advanced approaches could significantly improve the specificity, sensitivity, and versatility of tools for SPAC977.05c detection in complex research applications.

What approaches could integrate SPAC977.05c antibody detection with emerging single-cell analysis technologies?

Integration with single-cell technologies represents an important frontier:

Integration strategies:

• Single-cell western blot adaptation:

  • Microfluidic single-cell western blot

  • Capillary electrophoresis with immunodetection

  • Miniaturized gel electrophoresis systems

• Mass cytometry approaches:

  • Metal-labeled SPAC977.05c antibodies for CyTOF

  • Integration with cell cycle markers and other proteins

  • Development of balanced panel design

• Spatial proteomics integration:

  • Adaptation for Imaging Mass Cytometry

  • Integration with CODEX multiplexed imaging

  • Development of clearing protocols compatible with antibody retention

• Single-cell sequencing integration:

  • CITE-seq adaptation (cellular indexing of transcriptomes and epitopes)

  • Antibody-oligonucleotide conjugates for REAP-seq

  • Development of combined protein and transcriptome profiling