SPAC1093.07 Antibody

Basic Research Questions

• What is SPAC1093.07 and why would researchers use antibodies against it?

  SPAC1093.07 is a protein-coding gene in Schizosaccharomyces pombe (fission yeast), classified as a "dubious" or hypothetical protein . Researchers use antibodies against it primarily for studying protein expression patterns in S. pombe, particularly in cell cycle research and fungal protein interaction studies. The protein is often investigated in conjunction with other fission yeast proteins such as those involved in actin binding or cell cortex formations . Methodologically, these antibodies serve as valuable tools for identifying protein localization through immunofluorescence and protein expression through Western blotting, helping researchers understand the functional role of SPAC1093.07 in yeast cellular processes.

• What applications has the SPAC1093.07 Antibody been validated for?

  The SPAC1093.07 Antibody has been validated for multiple research applications including:

  • ELISA (Enzyme-Linked Immunosorbent Assay)

  • Western Blot (WB)

  When using this antibody for Western blot applications, researchers should optimize dilution ratios based on sample concentration. The antibody has been antigen-affinity purified, which enhances specificity for the target protein . For researchers planning immunofluorescence studies, it's recommended to perform preliminary optimization experiments with positive controls from S. pombe lysates, similar to protocols established for other yeast proteins like Pma1 .

• What are the optimal storage and handling conditions for SPAC1093.07 Antibody?

  For maximum stability and performance of the SPAC1093.07 Antibody, follow these guidelines:

  • Store at -20°C or -80°C upon receipt

  • Avoid repeated freeze-thaw cycles

  • The antibody is supplied in 50% glycerol with 0.03% Proclin 300 and 0.01M PBS at pH 7.4

  • Do not freeze the antibody, as mentioned in source documentation

  For long-term storage projects, aliquoting the antibody into smaller volumes before freezing is recommended to avoid degradation from repeated freeze-thaw cycles. Unlike some antibodies that can be stored at 2-8°C for short periods, this particular antibody requires freezer storage for maintaining its binding capacity.

• What controls should be included when working with SPAC1093.07 Antibody?

  When designing experiments with SPAC1093.07 Antibody, include these essential controls:

  1. Positive control: Lysate from wild-type S. pombe strain 972

  2. Negative control:

     • Primary antibody omission

     • Lysate from SPAC1093.07 knockout strain (if available)

  3. Loading control: Use antibodies against conserved proteins like Pma1, which serves as an established membrane protein marker in yeast

  For immunofluorescence experiments, include a secondary-only control to assess non-specific binding. These controls are critical for validating experimental results and distinguishing specific from non-specific signals, especially since SPAC1093.07 is classified as a hypothetical protein.

Advanced Research Questions

• How can researchers optimize Western blot protocols for SPAC1093.07 Antibody?

  Optimizing Western blot protocols for SPAC1093.07 Antibody requires several key adjustments:

  1. Sample preparation:

     • Use glass bead lysis in 50mM Tris-HCl pH 7.5, 150mM NaCl, 5mM EDTA, 10% glycerol with protease inhibitors

     • Heat samples at 65°C rather than 95°C to prevent aggregation of membrane proteins

  2. Gel electrophoresis and transfer:

     • Use 10-12% SDS-PAGE gels for optimal resolution

     • Transfer to PVDF membrane at 30V overnight at 4°C for complete transfer

  3. Antibody incubation:

     • Block with 5% non-fat dry milk in TBS-T for 1 hour

     • Dilute primary antibody 1:1000 to 1:2000 in blocking buffer

     • Incubate overnight at 4°C with gentle agitation

  4. Detection optimization:

     • Use enhanced chemiluminescence (ECL) detection with exposure times of 30 seconds to 5 minutes

     • Consider using signal enhancers if the target protein has low expression levels

  This protocol is similar to those used for other S. pombe proteins and should be further optimized based on laboratory-specific conditions.

• What experimental approaches can verify SPAC1093.07 Antibody specificity?

  Validating SPAC1093.07 Antibody specificity requires multiple complementary approaches:

  1. Genetic validation:

     • Compare antibody reactivity between wild-type and SPAC1093.07 deletion strains

     • Use CRISPR-Cas9 gene editing to introduce epitope tags for parallel detection

  2. Biochemical validation:

     • Pre-absorb antibody with recombinant SPAC1093.07 protein before immunostaining

     • Perform peptide competition assays using the immunogen peptide

  3. Cross-reactivity assessment:

     • Test against related S. pombe proteins

     • Express SPAC1093.07 in heterologous systems (E. coli, mammalian cells)

  4. Mass spectrometry validation:

     • Immunoprecipitate using the antibody and analyze by mass spectrometry

     • Compare detected proteins with expected size and sequence

  These validation steps are particularly important for hypothetical proteins like SPAC1093.07 where functional characterization is limited.

• How does SPAC1093.07 protein expression change during the S. pombe cell cycle?

  Monitoring SPAC1093.07 protein expression throughout the cell cycle requires synchronized cultures and multiple detection points:

  1. Synchronization methods:

     • Temperature-sensitive cdc mutants

     • Nitrogen starvation-release

     • Lactose gradient centrifugation

  2. Sampling protocol:

     • Collect samples every 20 minutes for 4 hours

     • Process for both protein extraction (Western blotting) and fixation (immunofluorescence)

  3. Cell cycle markers:

     • DNA content (flow cytometry with propidium iodide)

     • Septation index (aniline blue staining)

     • Nuclear position (DAPI staining)

  4. Expression analysis:

     • Normalize SPAC1093.07 signal to loading controls

     • Plot expression relative to cell cycle markers

  This comprehensive approach will reveal whether SPAC1093.07 shows cell cycle-dependent expression patterns, providing insights into its potential cellular functions.

• Can SPAC1093.07 Antibody be used for co-immunoprecipitation experiments?

  Using SPAC1093.07 Antibody for co-immunoprecipitation (co-IP) requires:

  1. Optimization strategy:

     • Test different lysis conditions (varying detergents: NP-40, Triton X-100, CHAPS)

     • Compare direct antibody coupling vs. protein A/G beads

     • Use mild washing conditions to preserve protein-protein interactions

  2. Protocol outline:

     • Lyse cells in 50mM HEPES pH 7.5, 150mM NaCl, 1mM EDTA, 1% NP-40 with protease inhibitors

     • Pre-clear lysate with protein A/G beads for 1 hour

     • Incubate with SPAC1093.07 Antibody (5-10 μg) overnight at 4°C

     • Add protein A/G beads for 2 hours

     • Wash 4-5 times with lysis buffer

     • Elute with SDS sample buffer or low pH buffer

  3. Controls:

     • IgG control precipitation

     • Immunoprecipitation from SPAC1093.07 knockout strain

  Co-IP experiments can identify interaction partners of SPAC1093.07, potentially revealing its functional networks within the fission yeast proteome.

• How can SPAC1093.07 Antibody be integrated into multi-color immunofluorescence experiments?

  For multi-color immunofluorescence incorporating SPAC1093.07 Antibody:

  1. Antibody combination strategy:

     • Pair rabbit polyclonal SPAC1093.07 Antibody with mouse monoclonal antibodies against other targets

     • Use appropriate secondary antibodies with non-overlapping emission spectra

  2. Sample preparation:

     • Fix cells with 3.7% formaldehyde for 30 minutes

     • Permeabilize with 1% Triton X-100 for 2 minutes

     • Block with 5% BSA in PBS for 1 hour

  3. Staining protocol:

     • Apply primary antibodies sequentially or simultaneously (after testing for interference)

     • Include nuclear stain (DAPI) and cell wall stain (aniline blue)

     • Mount with anti-fade mounting medium

  4. Imaging considerations:

     • Use sequential scanning to minimize bleed-through

     • Include single-stained controls for each fluorophore

     • Perform colocalization analysis using appropriate software

  This approach allows researchers to examine SPAC1093.07 localization in relation to other cellular compartments or proteins of interest.

• What are the limitations of using polyclonal antibodies against SPAC1093.07?

  Researchers should be aware of these limitations when working with polyclonal SPAC1093.07 Antibody:

  1. Batch-to-batch variability:

     • Each antibody lot may have different epitope recognition profiles

     • Perform validation with each new lot

  2. Potential cross-reactivity:

     • May recognize related proteins in S. pombe or other yeasts

     • Could detect post-translationally modified forms differently

  3. Background considerations:

     • Higher background in certain applications compared to monoclonals

     • May require additional blocking steps (5% BSA + 5% normal serum)

  4. Application constraints:

     • May not perform equally well across all applications

     • Some polyclonals work better for Western blot than immunofluorescence

  Researchers should document the specific lot number used for experiments to enhance reproducibility, especially for long-term projects studying this hypothetical protein.

• How does sample preparation affect SPAC1093.07 detection in fission yeast?

  Sample preparation significantly impacts SPAC1093.07 detection:

  Preparation Method	Advantages	Limitations	Recommended For
  TCA precipitation	High protein recovery	Harsh conditions may alter epitopes	Western blotting
  Spheroplasting	Preserves protein complexes	Time-consuming	Co-immunoprecipitation
  Mechanical disruption	Simple, fast	May cause protein degradation	Quick screening
  Chemical lysis	Good for membrane proteins	May not extract all proteins	Studying membrane-associated forms

  For optimal results with SPAC1093.07 Antibody:

  1. Include protease and phosphatase inhibitors regardless of method

  2. Process samples quickly and keep cold throughout

  3. Use gentle extraction methods if studying protein-protein interactions

  4. Consider specialized extraction methods if SPAC1093.07 has membrane associations

  The choice of sample preparation should align with the specific research question and downstream application.

• How can researchers troubleshoot non-specific binding with SPAC1093.07 Antibody?

  When encountering non-specific binding with SPAC1093.07 Antibody, implement this systematic troubleshooting approach:

  1. Optimize blocking conditions:

     • Try different blocking agents (BSA, non-fat dry milk, normal serum)

     • Increase blocking time (overnight at 4°C)

     • Add 0.1% Tween-20 to reduce hydrophobic interactions

  2. Adjust antibody parameters:

     • Titrate antibody concentration (try 1:500 to 1:5000 dilutions)

     • Reduce incubation time or temperature

     • Use antibody diluent with protein carriers

  3. Modify washing protocol:

     • Increase number and duration of washes

     • Use higher salt concentration in wash buffer (up to 500mM NaCl)

     • Add 0.1% SDS to wash buffer for Western blots

  4. Sample-specific adjustments:

     • Pre-absorb antibody with non-specific proteins (E. coli lysate)

     • Use alternative fixation methods for immunofluorescence

     • Include competing peptides to verify specific binding

  Document all optimization steps methodically to establish a reliable protocol for future experiments.

• What approaches can distinguish between SPAC1093.07 and other closely related proteins?

  To distinguish SPAC1093.07 from related proteins:

  1. Genetic approaches:

     • Generate strain-specific knockouts as negative controls

     • Create epitope-tagged versions for parallel detection

     • Use CRISPR-Cas9 to introduce mutations in potential cross-reactive proteins

  2. Analytical techniques:

     • Perform 2D gel electrophoresis followed by Western blotting

     • Use super-resolution microscopy to compare localization patterns

     • Employ mass spectrometry to verify protein identity

  3. Immunological methods:

     • Develop peptide-specific antibodies targeting unique regions

     • Perform peptide competition assays with fragments from SPAC1093.07 and related proteins

     • Use epitope mapping to determine exact binding sites

  4. Bioinformatic analysis:

     • Identify unique peptide sequences through sequence alignment

     • Target these regions for future antibody development

     • Use structural prediction to identify exposed epitopes

  These approaches help ensure experimental results are specifically attributed to SPAC1093.07 rather than related proteins.

• How can researchers quantitatively assess SPAC1093.07 Antibody binding characteristics?

  For quantitative assessment of SPAC1093.07 Antibody binding:

  1. Surface Plasmon Resonance (SPR) analysis:

     • Determine kon and koff rates

     • Calculate equilibrium dissociation constant (KD)

     • Compare affinity across different conditions

  2. Quantitative ELISA:

     • Generate standard curves with recombinant protein

     • Determine linear range for quantification

     • Calculate antibody EC50 values

  3. Dose-response experiments:

     • Titrate antibody concentrations (0.1-10 μg/ml)

     • Plot binding curves and calculate saturation points

     • Determine optimal working concentration

  4. Competition binding assays:

     • Use labeled and unlabeled antibody

     • Calculate IC50 values for binding inhibition

     • Determine epitope accessibility

  These quantitative approaches provide rigorous characterization of antibody performance, essential for reproducible research and method standardization.

• What experimental strategies integrate SPAC1093.07 Antibody with genetic approaches in S. pombe research?

  Integrating antibody-based detection with genetic approaches provides powerful insights:

  1. Conditional expression systems:

     • Use nmt1 promoter to control SPAC1093.07 expression

     • Monitor protein levels with antibody during induction/repression

     • Correlate expression with phenotypic changes

  2. Mutation analysis:

     • Create point mutations in SPAC1093.07

     • Use antibody to detect expression/stability changes

     • Determine structure-function relationships

  3. Tagged protein complementation:

     • Express tagged versions in SPAC1093.07 deletion background

     • Use both tag antibodies and SPAC1093.07 Antibody

     • Verify functional complementation

  4. Synthetic genetic arrays:

     • Cross SPAC1093.07 mutants with genome-wide deletion library

     • Use antibody to verify protein status in genetic interactions

     • Identify functional pathways

  This integrated approach combines the specificity of genetic manipulation with the detection power of SPAC1093.07 Antibody to elucidate protein function.