SPBC21B10.07 Antibody

What is SPBC21B10.07 and why is it important in research?

SPBC21B10.07 is a gene/protein from Schizosaccharomyces pombe (fission yeast), identifiable in databases such as KEGG (spo:SPBC21B10.07) and STRING (4896.SPBC21B10.07.1) . S. pombe serves as an important model organism for studying fundamental eukaryotic cellular processes, including cell cycle regulation, DNA repair mechanisms, and chromosome dynamics. The protein encoded by SPBC21B10.07 is of particular interest for researchers investigating conserved cellular pathways that may have homologs in higher eukaryotes including humans.

The antibody against this protein provides a critical research tool for:

• Determining subcellular localization

• Studying protein expression levels under different conditions

• Investigating protein-protein interactions

• Analyzing post-translational modifications

What validation techniques should be used for SPBC21B10.07 antibodies?

Proper validation of SPBC21B10.07 antibodies is essential for generating reliable experimental data. A multi-faceted approach is recommended:

What are the optimal storage conditions for SPBC21B10.07 antibodies?

To maintain antibody functionality and prevent degradation, SPBC21B10.07 antibodies should be stored according to established guidelines:

• Store at 2-8°C for up to 12 months for conjugated antibodies

• For long-term storage, aliquot and maintain at -20°C to -80°C

• Avoid repeated freeze-thaw cycles (limit to <5)

• Protect fluorophore-conjugated antibodies from light exposure

• Do not freeze certain conjugated antibodies (follow manufacturer specifications)

How should experiments be designed to study SPBC21B10.07 protein interactions?

When investigating SPBC21B10.07 protein interactions, consider implementing these methodological approaches:

• Co-immunoprecipitation (Co-IP): Use anti-SPBC21B10.07 antibodies to pull down the protein complex, followed by Western blotting for potential interacting partners. Include appropriate controls:

  • IgG control precipitation

  • Reverse Co-IP with antibodies against suspected interacting partners

  • Knockout/knockdown validation

• Proximity Ligation Assay (PLA): This technique can visualize protein-protein interactions in situ with high sensitivity.

  • Use primary antibodies from different species against SPBC21B10.07 and potential interacting partners

  • Verify antibody compatibility in multiplex assays

  • Include positive and negative interaction controls

• Bimolecular Fluorescence Complementation (BiFC): For live-cell interaction studies.

The experimental approach should incorporate appropriate controls and statistical design principles as outlined in standard reference texts for biological research .

What are the optimal conditions for Western blotting with SPBC21B10.07 antibodies?

Optimized Western blot protocols for SPBC21B10.07 antibodies should consider:

Sample Preparation:

• Lyse cells in buffer containing protease inhibitors

• Include phosphatase inhibitors if studying phosphorylation states

• Determine optimal protein load (typically 20-50 μg total protein)

Electrophoresis and Transfer:

• Use appropriate percentage acrylamide gel based on protein size

• Optimize transfer conditions (wet or semi-dry) based on protein size

Antibody Incubation:

• Test antibody dilutions (typically starting at 1:1000)

• Determine optimal blocking solution (5% BSA or non-fat milk)

• Optimize incubation time and temperature (4°C overnight or room temperature for 1-2 hours)

Detection:

• Select appropriate secondary antibody (species-specific, conjugated to HRP or fluorophore)

• Include molecular weight markers

• Consider using loading controls for normalization

Similar methodological considerations should be applied as those used in flow cytometry applications for other research antibodies .

How should immunofluorescence experiments with SPBC21B10.07 antibodies be designed?

Effective immunofluorescence with SPBC21B10.07 antibodies requires:

Fixation Optimization:

• Test multiple fixation methods (paraformaldehyde, methanol, or acetone)

• Optimize fixation time and temperature

• Evaluate permeabilization conditions (Triton X-100, saponin, or digitonin)

Antibody Parameters:

• Titrate primary antibody concentrations

• Optimize incubation conditions (time, temperature, buffer composition)

• Select appropriate fluorophore-conjugated secondary antibodies

Controls:

• Include negative controls (secondary antibody only, isotype control)

• Use knockout/knockdown samples as specificity controls

• Include positive controls with known localization patterns

Image Acquisition:

• Use appropriate microscopy technique (confocal, wide-field, super-resolution)

• Standardize exposure settings across samples

• Collect z-stacks for 3D localization analysis

How should Western blot data with SPBC21B10.07 antibodies be quantified?

Accurate quantification of Western blot data requires:

• Image acquisition using a linear detection system (digital imager preferred over film)

• Analysis with specialized software (ImageJ, Image Studio, etc.)

• Background subtraction using appropriate methods

• Normalization to loading controls (GAPDH, β-actin, total protein stain)

• Statistical analysis across multiple biological replicates

Quantification Protocol:

• Define regions of interest (ROIs) of consistent size

• Subtract local background from each lane

• Normalize to loading control

• Calculate relative expression compared to control condition

• Perform statistical analysis (t-test, ANOVA) as appropriate

How can cross-reactivity issues be identified and resolved in SPBC21B10.07 antibody studies?

Cross-reactivity presents a significant challenge in antibody-based research. For SPBC21B10.07 studies:

Identification of Cross-Reactivity:

• Unexpected bands in Western blots

• Non-specific staining patterns in immunofluorescence

• Positive signals in knockout/knockdown controls

• Inconsistent results across different antibody lots

Resolution Strategies:

• Test multiple antibodies targeting different epitopes

• Use antibody combination approaches, similar to strategies employed for challenging targets such as SARS-CoV-2 variants

• Implement epitope mapping to identify specificity determinants

• Adjust blocking conditions (increase BSA concentration, add mild detergents)

• Pre-absorb antibody with related proteins or lysates from knockout cells

How can SPBC21B10.07 post-translational modifications be studied using antibody-based approaches?

Investigating post-translational modifications (PTMs) of SPBC21B10.07 requires specialized approaches:

Phosphorylation Studies:

• Use phospho-specific antibodies if available

• Combine with phosphatase treatments as controls

• Consider Phos-tag gels to separate phosphorylated species

• Use mass spectrometry to identify specific phosphorylation sites

Ubiquitination Analysis:

• Perform immunoprecipitation under denaturing conditions

• Blot for ubiquitin after SPBC21B10.07 immunoprecipitation

• Use proteasome inhibitors to stabilize ubiquitinated proteins

SUMOylation Detection:

• Similar to ubiquitination analysis with SUMO-specific antibodies

• Consider temperature-sensitive SUMO protease mutants for validation

Methodological Considerations:

• Include appropriate positive controls for each PTM

• Validate PTM-specific antibodies rigorously

• Consider genetic approaches (mutate PTM sites) for validation

How do SPBC21B10.07 variants affect antibody recognition in different S. pombe strains?

Genetic variations can significantly impact antibody epitope recognition, similar to observations with SARS-CoV-2 variant studies :

Analysis Approach:

• Sequence SPBC21B10.07 across different strains to identify variations

• Map variations to known antibody epitopes

• Test antibody binding using recombinant proteins with specific mutations

• Develop antibody combinations targeting conserved epitopes

Mitigation Strategies:

• Use multiple antibodies targeting different epitopes

• Design new antibodies against highly conserved regions

• Consider aptamer-based alternatives for highly variable regions

• Implement computational prediction of epitope conservation

Studies of antibody escape in viral variants provide instructive parallels, where single mutations can significantly reduce antibody recognition .

How can multiplexed detection systems incorporating SPBC21B10.07 antibodies be developed?

Developing multiplexed systems for simultaneous detection of SPBC21B10.07 and other targets:

Methodological Approaches:

• Flow Cytometry Multiplexing:

  • Select non-overlapping fluorophores for each antibody

  • Conduct titration experiments for each antibody individually

  • Test antibody combinations for interference effects

  • Include appropriate compensation controls

• Multiplex Immunofluorescence Imaging:

  • Use spectral unmixing for closely overlapping fluorophores

  • Implement sequential staining protocols if antibody species conflict

  • Consider tyramide signal amplification for low-abundance targets

  • Use nuclear counterstains for cell identification

• Multiplex Western Blotting:

  • Utilize different fluorophores for simultaneous detection

  • Implement stripping and re-probing protocols for sequential detection

  • Validate that stripping does not affect subsequent antibody binding

Careful validation using single-plex controls is essential for accurate interpretation of multiplex data.

What are common sources of variability in SPBC21B10.07 antibody experiments?

Understanding sources of variability is crucial for reproducible research:

Implementing standard operating procedures (SOPs) and quality control checkpoints can significantly reduce experimental variability.

How should contradictory results with different SPBC21B10.07 antibodies be reconciled?

When different antibodies targeting SPBC21B10.07 yield contradictory results:

• Verify epitope differences:

  • Map the epitopes recognized by each antibody

  • Determine if post-translational modifications affect recognition

  • Consider if protein conformation impacts antibody accessibility

• Validate each antibody independently:

  • Test specificity using knockout/knockdown controls

  • Perform peptide competition assays

  • Evaluate performance across multiple applications

• Consider biological explanations:

  • Different isoforms or splice variants

  • Stage-specific or condition-specific modifications

  • Protein-protein interactions masking epitopes

• Implement orthogonal techniques:

  • Use non-antibody methods (MS, CRISPR tagging)

  • Apply genetic approaches (mutational analysis)

  • Consider in vitro binding studies with recombinant proteins

Similar approaches have been used to reconcile contradictory findings in studies of antibody responses to viral variants .