SPAC1327.01c Antibody

What is SPAC1327.01c protein and what cellular processes is it involved in?

SPAC1327.01c (Uniprot No. Q1MTM9) is a protein expressed in Schizosaccharomyces pombe (fission yeast, strain 972/ATCC 24843). While the search results don't specify its exact function, antibodies against such yeast proteins are typically used in fundamental research to study protein expression, localization, and function in cellular pathways. Similar research approaches on bacterial proteins have proven valuable, as seen with Staphylococcus aureus Protein A studies . For SPAC1327.01c research, investigators should first establish baseline expression patterns using the antibody in wild-type yeast before proceeding to genetic manipulation experiments.

What are the optimal storage conditions for maintaining SPAC1327.01c antibody activity?

SPAC1327.01c antibody should be stored at -20°C or -80°C upon receipt and researchers should avoid repeated freeze-thaw cycles that could compromise antibody function . The antibody is supplied in liquid form with a storage buffer containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4 . This formulation helps maintain stability during long-term storage. For working aliquots, researchers should divide the stock into small volumes to minimize freeze-thaw cycles, similar to protocols used with other research antibodies like M0313, which maintained activity when properly stored .

What validation methods confirm the specificity of SPAC1327.01c antibody?

The SPAC1327.01c antibody has been validated for ELISA and Western Blot applications to ensure antigen identification specificity . When establishing a new research protocol, validation should include:

• Western blot analysis comparing wild-type and knockout strains

• Immunoprecipitation followed by mass spectrometry

• Comparative analysis with multiple antibody lots if available

These validation approaches mirror successful strategies employed with other research antibodies, such as the anti-SpA5 antibody Abs-9, which underwent rigorous characterization prior to experimental application .

What is the recommended protocol for using SPAC1327.01c antibody in Western blotting?

For Western blot applications with SPAC1327.01c antibody, researchers should:

• Prepare yeast protein extracts using mechanical disruption (glass beads) or enzymatic methods (zymolyase treatment)

• Separate proteins using SDS-PAGE (10-12% gel recommended for most yeast proteins)

• Transfer proteins to PVDF or nitrocellulose membrane

• Block with 5% non-fat milk or BSA in TBST

• Incubate with SPAC1327.01c antibody (recommended starting dilution: 1:1000, then optimize)

• Wash thoroughly with TBST

• Incubate with appropriate secondary antibody (anti-rabbit IgG-HRP)

• Develop using chemiluminescence detection

This protocol draws on established practices similar to those used with other polyclonal antibodies targeting microbial proteins, such as antibodies against Staphylococcal proteins, which have been successfully employed in research settings .

How can SPAC1327.01c antibody be optimized for immunofluorescence microscopy in yeast cells?

For immunofluorescence applications with fission yeast:

• Fix cells with 3.7% formaldehyde for 30 minutes

• Digest cell wall with zymolyase (1mg/ml) for 30 minutes

• Permeabilize with 0.5% Triton X-100

• Block with 1% BSA in PBS

• Incubate with SPAC1327.01c antibody (starting dilution 1:200)

• Wash thoroughly with PBS

• Incubate with fluorophore-conjugated anti-rabbit secondary antibody

• Counterstain nuclei with DAPI

• Mount and visualize

Researchers should include appropriate controls, including secondary antibody-only controls and pre-immune serum controls. This methodology builds upon immunolocalization techniques that have successfully determined protein distribution patterns in similar experimental systems .

What cross-reactivity concerns should researchers address when using SPAC1327.01c antibody?

As a polyclonal antibody raised against recombinant SPAC1327.01c protein, potential cross-reactivity with structurally similar proteins should be carefully evaluated . Researchers should:

• Perform bioinformatic analysis to identify proteins with sequence homology

• Include knockout/knockdown controls where possible

• Pre-absorb the antibody with recombinant protein when high specificity is required

• Compare staining patterns with alternative detection methods

Similar cross-reactivity assessments proved essential in characterizing antibodies like M0313, where epitope mapping confirmed specificity against the target protein .

How can epitope mapping be performed to characterize SPAC1327.01c antibody binding sites?

For researchers seeking to identify specific binding epitopes of SPAC1327.01c antibody:

• Generate a peptide library spanning the full SPAC1327.01c sequence (typically 15-20 amino acid peptides with 5-10 amino acid overlaps)

• Screen peptides by ELISA using the SPAC1327.01c antibody

• Confirm positive hits with competitive binding assays

• Refine epitope identification using alanine scanning mutagenesis

• Validate findings using in silico molecular docking approaches similar to those applied with Abs-9 antibody

This comprehensive epitope mapping strategy provides insights into antibody functionality and can guide the design of improved immunological tools, as demonstrated with other antibodies targeting microbial proteins .

What strategies enable quantification of SPAC1327.01c protein expression levels across different experimental conditions?

For precise quantification of SPAC1327.01c expression:

• Establish a calibration curve using purified recombinant SPAC1327.01c protein

• Develop a quantitative ELISA using the SPAC1327.01c antibody

• Implement quantitative Western blotting with:

  • Internal loading controls (e.g., actin)

  • Chemiluminescence detection with linear dynamic range

  • Image analysis software for densitometry

• Consider complementary approaches:

  • qRT-PCR for mRNA expression correlation

  • Mass spectrometry-based absolute quantification

This multi-faceted approach mirrors quantification strategies successfully applied in other protein expression studies, providing robust data for comparative analyses across experimental conditions .

How can researchers employ SPAC1327.01c antibody for chromatin immunoprecipitation (ChIP) experiments?

For adapting SPAC1327.01c antibody to ChIP applications:

• Cross-link S. pombe cells with 1% formaldehyde for 15 minutes

• Lyse cells and sonicate chromatin to 200-500bp fragments

• Pre-clear lysate with protein A/G beads

• Immunoprecipitate with SPAC1327.01c antibody (5-10μg per reaction)

• Include appropriate controls:

  • IgG control

  • Input chromatin

  • Immunoprecipitation with antibody against known DNA-associated proteins

• Wash stringently to remove non-specific interactions

• Reverse cross-links and purify DNA

• Analyze by qPCR or next-generation sequencing

This protocol adaptation draws on established ChIP methodologies while accounting for the specific characteristics of fission yeast chromatin and the SPAC1327.01c antibody properties .

How should researchers address non-specific background when using SPAC1327.01c antibody in immunoassays?

When encountering high background signal:

• Optimize blocking conditions:

  • Test alternative blocking agents (BSA, casein, commercial blockers)

  • Increase blocking time and concentration

• Adjust antibody parameters:

  • Titrate primary antibody concentration

  • Reduce incubation time or temperature

  • Include 0.1-0.5% detergent in antibody diluent

• Implement additional controls:

  • Pre-absorb antibody with recombinant antigen

  • Compare signal in wild-type vs. knockout samples

• Modify washing procedures:

  • Increase number and duration of washes

  • Use higher stringency wash buffers

These optimization strategies have proven effective in reducing background interference, as demonstrated in studies with other research antibodies targeting microbial proteins .

What factors influence variability in SPAC1327.01c detection between experiments?

To address inter-experimental variability, researchers should consider:

• Antibody-related factors:

  • Lot-to-lot variation (request COA for each lot)

  • Storage conditions and freeze-thaw history

  • Working dilution optimization for each new lot

• Sample preparation variables:

  • Consistency in growth conditions for S. pombe

  • Standardized protein extraction protocols

  • Protein quantification accuracy

• Assay standardization:

  • Include positive controls in each experiment

  • Implement internal reference standards

  • Document all protocol deviations

Rigorous attention to these variables significantly improves reproducibility, as demonstrated in comprehensive antibody characterization studies like those conducted for the SC27 and Abs-9 antibodies .

How can researchers conclusively differentiate between specific and non-specific signals when analyzing complex data sets?

For robust differentiation between specific and non-specific signals:

• Implement a multi-tiered validation approach:

  • Genetic controls (knockout/knockdown)

  • Competitive inhibition with recombinant protein

  • Signal correlation across multiple detection methods

• Apply quantitative analysis:

  • Signal-to-noise ratio determination

  • Statistical assessment of signal distribution

  • Bayesian analysis for probability of true positive signals

• Utilize orthogonal detection methods:

  • Mass spectrometry validation of immunoprecipitated proteins

  • Correlation between protein and mRNA levels

  • Alternative antibodies targeting different epitopes

This comprehensive validation strategy ensures data reliability similar to the approach used for characterizing the M0313 antibody, where multiple independent methods confirmed target specificity .

How might high-throughput approaches accelerate research using SPAC1327.01c antibody?

Researchers can leverage high-throughput methodologies by:

• Adapting SPAC1327.01c antibody for microarray applications:

  • Protein microarrays for interaction studies

  • Antibody microarrays for expression profiling

  • Tissue microarrays for localization studies

• Implementing automated immunoassay platforms:

  • Robotics-assisted Western blotting

  • High-content imaging systems

  • Automated ELISA workflows

• Integrating with -omics approaches:

  • Correlation with transcriptomics data

  • Integration with proteomics datasets

  • Network analysis with interactome data

These approaches parallel successful high-throughput antibody applications demonstrated in recent studies, such as the identification of anti-SpA5 antibodies using high-throughput single-cell RNA and VDJ sequencing .

What considerations are important when designing experiments combining SPAC1327.01c antibody with CRISPR-Cas9 gene editing?

For integrating SPAC1327.01c antibody with CRISPR-Cas9 approaches:

• Experimental design considerations:

  • Validate antibody specificity pre-editing

  • Design appropriate sgRNA controls

  • Include wild-type controls alongside edited cells

• Analytical framework:

  • Quantitative assessment of protein level changes

  • Correlation between editing efficiency and protein depletion

  • Time-course analysis of protein turnover post-editing

• Potential pitfalls and solutions:

  • Truncated proteins may retain epitopes — evaluate with multiple antibodies

  • Compensatory expression of related proteins — perform pathway analysis

  • Off-target effects — implement comprehensive validation strategies

This integrative approach ensures meaningful data interpretation when combining antibody-based detection with gene editing technologies, similar to strategies employed in other protein-targeting studies .

How can computational approaches enhance the utility of SPAC1327.01c antibody in structural biology research?

Computational methods can significantly extend antibody applications:

• Epitope prediction and modeling:

  • In silico epitope mapping

  • Molecular docking simulations

  • Ab initio modeling of antibody-antigen complexes

• Structure-function relationship analysis:

  • Homology modeling of SPAC1327.01c protein

  • Prediction of functional domains

  • Identification of potentially important interaction sites

• Integration with experimental data:

  • Refinement of computational models with experimental binding data

  • Virtual screening for potential cross-reactive proteins

  • Simulation of binding kinetics

These computational approaches have demonstrated value in antibody research, as evidenced by the successful epitope prediction for Abs-9 using AlphaFold2 and molecular docking methods .