SPCC613.03 Antibody

Basic Research Questions

• What is the SPCC613.03 Antibody and what organism is it specific to?

  SPCC613.03 Antibody (product code CSB-PA529219XA01SXV, UniProt number O74903) is a polyclonal antibody raised against recombinant proteins from Schizosaccharomyces pombe (strain 972 / ATCC 24843), commonly known as fission yeast . The antibody is designed to recognize specific epitopes of the SPCC613.03 protein, which plays roles in cellular functions within S. pombe. Unlike antibodies raised against mammalian targets, this antibody is specifically developed for research involving fission yeast models, which are extensively used to study eukaryotic cell cycle regulation, DNA damage responses, and cellular morphogenesis.

• What applications is the SPCC613.03 Antibody validated for?

  Based on typical validation profiles for S. pombe antibodies, the SPCC613.03 Antibody has been tested and validated for applications including Western blot (WB) and ELISA . When using this antibody for immunocytochemistry (ICC) or other applications, researchers should conduct preliminary validation experiments since antibodies that perform well in Western blotting do not necessarily show equivalent specificity in ICC or immunofluorescence applications . Research findings indicate that application-specific validation is critical, as the performance of antibodies can vary significantly between assays even when using the same epitope recognition principle.

• What is the recommended storage and handling protocol for SPCC613.03 Antibody?

  For optimal preservation of antibody function, SPCC613.03 Antibody should be stored at -20°C or -80°C upon receipt and repeated freeze-thaw cycles should be avoided . The antibody is typically supplied in a liquid form with a preservation buffer containing 0.03% Proclin 300 and 50% Glycerol in 0.01M PBS at pH 7.4 . When working with the antibody, it should be kept on ice, and contamination should be prevented by using sterile technique. For long-term storage integrity, aliquoting the antibody into smaller volumes before freezing is recommended to minimize the detrimental effects of repeated freeze-thaw cycles on antibody performance and specificity.

• What controls should be included when using SPCC613.03 Antibody in experiments?

  When designing experiments with SPCC613.03 Antibody, several controls are essential: 1) Positive control: wild-type S. pombe cells expressing the target protein; 2) Negative control: S. pombe deletion mutants lacking the SPCC613.03 gene or cells where the gene has been silenced; 3) Secondary antibody-only control: to detect potential non-specific binding of the secondary detection system; 4) Isotype control: a non-specific antibody of the same isotype as SPCC613.03 Antibody to identify non-specific binding events . These controls are particularly important given that studies have demonstrated that many commercial antibodies may display cross-reactivity with unintended targets, which could lead to misinterpretation of experimental results.

Advanced Research Questions

• How can I validate the specificity of a new batch of SPCC613.03 Antibody for my research?

  Rigorous validation of each new batch of SPCC613.03 Antibody is essential to ensure experimental reproducibility. A comprehensive validation protocol should include:

  • Western blot analysis using positive (wild-type cells) and negative (gene deletion) controls

  • Peptide competition assay, where pre-incubation of the antibody with excess immunizing peptide should abolish specific signals

  • Cross-reactivity testing against closely related proteins in S. pombe

  • Immunoprecipitation followed by mass spectrometry to confirm target identity

  • For ICC applications, correlation of signal with known localization patterns and absence of signal in knockout strains

  Recent investigations into antibody specificity have shown that batch-to-batch variation can significantly impact experimental outcomes, making consistent validation crucial for maintaining research quality .

• What are the known cross-reactivity issues with SPCC613.03 Antibody and how can they be mitigated?

  While specific cross-reactivity data for SPCC613.03 Antibody is limited, research on similar antibodies suggests potential concerns. Polyclonal antibodies against S. pombe proteins may cross-react with conserved domains in related proteins. To mitigate these issues:

  1. Perform pre-adsorption experiments with recombinant proteins of closely related family members

  2. Use higher dilutions of antibody to reduce non-specific binding

  3. Optimize blocking conditions using different blockers (BSA, normal serum, casein)

  4. Implement stringent washing procedures to remove weakly bound antibodies

  5. Consider using immunodepletion techniques to remove cross-reactive antibodies

  Studies examining antibody specificity against transcription factors demonstrated that 5-10% of commercially available antibodies showed significant cross-reactivity with unintended targets, highlighting the importance of these validation approaches .

• How does epitope masking affect SPCC613.03 Antibody detection in different experimental contexts?

  Epitope masking can significantly impact detection efficiency of SPCC613.03 in various experimental settings. This phenomenon occurs when post-translational modifications, protein-protein interactions, or conformational changes obscure the antibody recognition site. Research on protein glycosylation in fission yeast demonstrates that extensive O-mannosylation can mask protein epitopes, affecting antibody recognition . To address epitope masking:

  • Consider alternative fixation methods that better preserve epitope accessibility

  • Test different antigen retrieval protocols for fixed samples

  • Use denaturing conditions for Western blot to expose hidden epitopes

  • For protein complexes, try crosslinking studies to determine if protein interactions affect antibody binding

  • Employ multiple antibodies recognizing different regions of the target protein

  Studies of N-glycosylation on unusual N-X-A sequons in S/T-rich regions have shown that these modifications can compete with O-mannosylation and affect epitope accessibility in S. pombe proteins .

• How can SPCC613.03 Antibody be used to investigate protein localization dynamics during cell cycle progression in S. pombe?

  For investigating protein localization during cell cycle progression:

  1. Synchronize S. pombe cultures using standard methods (nitrogen starvation, hydroxyurea block, or cdc25-22 temperature-sensitive mutants)

  2. Collect samples at defined time points across the cell cycle

  3. Process for immunofluorescence microscopy using SPCC613.03 Antibody

  4. Co-stain with cell cycle markers (e.g., Sid4 for spindle pole bodies)

  5. Use confocal microscopy with z-stack acquisition to capture the entire cell volume

  6. Quantify signal intensity and localization patterns using appropriate image analysis software

  This approach can reveal dynamic changes in protein localization similar to those observed for transcription factors like FoxA2/HNF-3β, which show differential nuclear localization patterns during developmental processes . Time-lapse imaging with fluorescently tagged secondary antibodies can further enhance the temporal resolution of these studies.

Methodological Considerations

• What is the recommended protocol for using SPCC613.03 Antibody in chromatin immunoprecipitation (ChIP) experiments?

  When adapting SPCC613.03 Antibody for ChIP applications in S. pombe:

  1. Cross-link cells with 1% formaldehyde for 15 minutes at room temperature

  2. Quench with 125 mM glycine for 5 minutes

  3. Harvest cells and disrupt cell wall using glass beads

  4. Sonicate chromatin to 200-500 bp fragments

  5. Pre-clear chromatin with protein A/G beads

  6. Immunoprecipitate with 5-10 μg SPCC613.03 Antibody overnight at 4°C

  7. Include IgG control and input samples

  8. Wash stringently to remove non-specific interactions

  9. Reverse cross-links and purify DNA

  10. Analyze by qPCR or sequencing

  Optimization of antibody concentration is critical, as studies with transcription factor antibodies have shown that non-specific binding increases at higher antibody concentrations, potentially leading to false-positive results .

• How can I troubleshoot weak or absent signals when using SPCC613.03 Antibody in Western blot analysis?

  When encountering weak or absent signals in Western blots:

  Potential Issue	Troubleshooting Approach
  Low protein expression	Increase protein loading; use enrichment techniques (e.g., immunoprecipitation)
  Inefficient protein transfer	Optimize transfer conditions; verify transfer efficiency with reversible stain
  Epitope destruction	Try different sample preparation methods; reduce boiling time; use alternative buffers
  Antibody concentration	Titrate antibody concentrations; try overnight incubation at 4°C
  Detection system sensitivity	Switch to more sensitive detection methods (ECL Prime, fluorescent secondary antibodies)
  Blocking interference	Test different blocking agents (milk vs. BSA); reduce blocking time
  Protein degradation	Add protease inhibitors; reduce sample processing time; keep samples cold

  Research has shown that even antibodies that mark specifically their target in Western blotting do not necessarily show specific immunoreactivity in ICC, requiring application-specific optimization .

• What are the best practices for quantifying relative protein expression using SPCC613.03 Antibody?

  For accurate protein quantification using SPCC613.03 Antibody:

  1. Establish a standard curve using purified recombinant target protein

  2. Ensure samples fall within the linear range of detection

  3. Always normalize to appropriate loading controls validated for S. pombe (e.g., α-tubulin, GAPDH)

  4. Include internal calibration controls on each blot

  5. Use digital image acquisition with appropriate exposure times to avoid saturation

  6. Apply statistical methods to assess significance of observed differences

  7. Report both biological and technical replicates (minimum n=3)

  Studies comparing different antibodies against the same target have demonstrated that quantification accuracy can vary significantly between antibodies and detection methods . When possible, corroborate findings using alternative methods like mass spectrometry.

• How should SPCC613.03 Antibody be validated for use in co-immunoprecipitation studies investigating protein-protein interactions?

  For co-immunoprecipitation validation:

  1. Perform reciprocal co-IPs with antibodies against suspected interaction partners

  2. Include appropriate negative controls (IgG, unrelated proteins)

  3. Validate specificity using SPCC613.03 deletion strains

  4. Test different lysis conditions to preserve interactions (non-ionic detergents vs. more stringent conditions)

  5. Consider cross-linking approaches for transient interactions

  6. Confirm interactions using alternative methods (yeast two-hybrid, proximity ligation assay)

  7. Use mass spectrometry to identify additional interaction partners

  Research on antibody-based detection of protein complexes demonstrates that the choice of buffer conditions can dramatically affect which interactions are preserved during immunoprecipitation . Optimizing these conditions is essential when investigating novel protein-protein interactions in S. pombe.

• What considerations should be made when using SPCC613.03 Antibody in fixed versus live cell imaging applications?

  When comparing fixed versus live cell applications:

  1. Fixed cell considerations:

     • Different fixation methods (paraformaldehyde, methanol, acetone) can differentially affect epitope accessibility

     • Permeabilization conditions must be optimized for nuclear proteins

     • Antigen retrieval may be necessary to expose masked epitopes

     • Signal-to-noise ratio can be enhanced through longer antibody incubation times

  2. Live cell considerations:

     • Consider using Fab fragments for better penetration

     • Microinjection of labeled antibodies may be necessary

     • Fluorescent labeling must not interfere with epitope recognition

     • Phototoxicity and bleaching must be minimized during imaging

  Studies comparing different immunofluorescence techniques have shown that the choice of fixation method can significantly impact the detection of nuclear antigens, with some epitopes being particularly sensitive to certain fixatives . Testing multiple fixation protocols is recommended for optimal results.