SPCC1739.08c Antibody

What is SPCC1739.08c and why is it studied in fission yeast research?

SPCC1739.08c is a gene in Schizosaccharomyces pombe (fission yeast) that has been cataloged in major biological databases including KEGG (spo:SPCC1739.08c) and STRING (4896.SPCC1739.08c.1) . Fission yeast serves as an excellent model organism for studying fundamental cellular processes due to its relatively simple genome and genetic tractability. Researchers investigate SPCC1739.08c to understand its role in cellular functions, particularly in relation to stress responses. Studies on fission yeast transcriptional responses, such as adaptation to hydrogen peroxide exposure, may provide insights into the function of this gene and its encoded protein .

What applications are most suitable for SPCC1739.08c antibody in basic research?

SPCC1739.08c antibody is primarily used for protein detection and localization studies. Similar to other research antibodies, it can be employed in applications such as immunofluorescence for cellular localization studies, western blotting for protein expression analysis, and immunoprecipitation for protein-protein interaction studies . When designing experiments, researchers should consider the antibody's specificity, sensitivity, and validated applications. For immunofluorescence applications specifically, proper controls should be included to ensure accurate interpretation of results, similar to procedures established for other monoclonal antibodies like Sp-40C .

How should SPCC1739.08c antibody be stored and handled to maintain optimal activity?

For optimal antibody performance, proper storage and handling are essential. Short-term storage at 4°C for up to two weeks is recommended for immediate use. For long-term storage, divide the antibody solution into small aliquots (no less than 20 μl) and store at -20°C or -80°C to avoid freeze-thaw cycles that can degrade antibody quality . Some researchers add an equal volume of glycerol as a cryoprotectant prior to freezing for increased stability. When working with the antibody, maintain sterile conditions and avoid contamination that could compromise experimental results .

How can I determine the optimal antibody concentration for SPCC1739.08c detection in my experimental system?

Determining the optimal concentration of SPCC1739.08c antibody requires systematic titration experiments. Start with a broad range of dilutions (e.g., 1:100, 1:500, 1:1000, 1:5000) in your application of interest. For each concentration, analyze signal-to-noise ratio, looking for the dilution that provides the strongest specific signal with minimal background. Similar to optimization procedures for other antibodies like the Human IL-8/CXCL8 antibody, validation should include both positive and negative controls . The optimal concentration will vary depending on your specific application (western blot, immunofluorescence, ELISA) and the abundance of your target protein. Document your optimization process thoroughly for reproducibility in future experiments.

What are the recommended validation methods to confirm SPCC1739.08c antibody specificity?

Thorough validation of SPCC1739.08c antibody specificity is crucial for reliable experimental results. A comprehensive validation approach should include:

• Genetic controls: Testing the antibody in wild-type versus SPCC1739.08c knockout or knockdown cells

• Peptide competition assays: Pre-incubating the antibody with the immunizing peptide to block specific binding

• Immunoblotting: Confirming a single band of the expected molecular weight

• Multiple antibody approach: Using different antibodies targeting distinct epitopes of the same protein

• Cross-reactivity testing: Evaluating potential cross-reactivity with related proteins

Similar to validation approaches used for therapeutic antibodies, these methods help establish confidence in the specificity of your antibody .

How can I optimize SPCC1739.08c antibody for use in immunofluorescence experiments?

For successful immunofluorescence experiments with SPCC1739.08c antibody, consider the following optimization strategies:

• Fixation method: Compare different fixatives (paraformaldehyde, methanol, acetone) to determine which best preserves epitope accessibility

• Permeabilization conditions: Test different detergents (Triton X-100, Tween-20, saponin) at various concentrations

• Blocking conditions: Optimize blocking buffer composition (BSA, normal serum, commercial blockers) to minimize background

• Antibody dilution: Perform serial dilutions to find the optimal concentration

• Incubation conditions: Test different incubation times and temperatures

• Detection system: Compare secondary antibodies or amplification systems for optimal signal-to-noise ratio

As recommended for the Sp-40C antibody, incorporating appropriate controls and detailed documentation of methods is essential for reproducibility .

How can epitope mapping be performed to characterize the binding site of SPCC1739.08c antibody?

Epitope mapping for SPCC1739.08c antibody can be performed using several complementary approaches:

• Peptide array analysis: Synthesize overlapping peptides spanning the SPCC1739.08c protein sequence and test antibody binding to identify the specific region recognized.

• Mutational analysis: Create point mutations or deletions in the SPCC1739.08c protein and assess changes in antibody binding. This approach is similar to the mutation analysis performed for SARS-CoV-2 spike protein to map neutralizing antibody epitopes .

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Compare deuterium uptake in the presence and absence of the antibody to identify regions protected by antibody binding.

• X-ray crystallography or cryo-EM: Determine the three-dimensional structure of the antibody-antigen complex at atomic resolution, similar to structural approaches used for characterizing therapeutic antibodies .

• Computational prediction: Use in silico methods to predict potential epitopes based on protein sequence and structure.

A comprehensive epitope mapping strategy typically combines multiple methods to build a complete picture of the antibody-antigen interaction.

What strategies can be employed to improve SPCC1739.08c antibody specificity for challenging applications?

When working with challenging applications where SPCC1739.08c antibody shows suboptimal specificity, consider these advanced approaches:

• Affinity purification: Purify the antibody using immobilized antigen to enrich for specific binding molecules.

• Fc engineering: Introduce modifications like N297A (as used in therapeutic antibodies) to reduce non-specific binding through Fc receptors .

• Cross-adsorption: Pre-incubate the antibody with lysates from cells lacking the target protein to remove antibodies that bind non-specifically.

• Recombinant antibody technology: Convert the antibody to recombinant format and engineer improved specificity through targeted mutations.

• Single-cell sequencing and expression: Isolate and sequence single B cells to identify the most specific antibody clones, similar to approaches used for isolating therapeutic antibodies from convalescent patients .

• Application-specific optimization: Develop customized protocols for each application, systematically testing different buffer compositions, blocking agents, and detection methods.

Each of these approaches requires rigorous validation to confirm improved specificity without compromising sensitivity.

How can SPCC1739.08c antibody be used in multiplexed imaging approaches?

Implementing SPCC1739.08c antibody in multiplexed imaging requires careful planning:

• Antibody conjugation strategies:

  • Direct conjugation to fluorophores with distinct spectral properties

  • Use of secondary antibodies from different species

  • Employment of click chemistry for site-specific labeling

• Multiplexing protocols:

  • Sequential staining with intermittent stripping or quenching

  • Spectral unmixing to resolve overlapping fluorophore emissions

  • Cyclic immunofluorescence (CycIF) for highly multiplexed imaging

• Data analysis approaches:

  Technique	Advantages	Limitations	Software Tools
  Traditional co-localization	Simple implementation	Limited to 4-5 markers	ImageJ, CellProfiler
  Spectral unmixing	Resolves overlapping spectra	Requires specialized equipment	Zeiss ZEN, Leica LAS X
  High-dimensional analysis	Captures complex relationships	Computationally intensive	histoCAT, Phenograph

• Validation strategies:

  • Single-color controls to establish specificity

  • Biological controls with known co-localization patterns

  • Comparison with alternative detection methods

This approach allows for simultaneous visualization of SPCC1739.08c with other proteins of interest, enabling complex spatial relationship analysis similar to approaches used in immunological research .

What are the most common causes of non-specific binding when using SPCC1739.08c antibody, and how can they be addressed?

Non-specific binding is a common challenge when working with antibodies. For SPCC1739.08c antibody, consider these potential issues and solutions:

• Insufficient blocking:

  • Problem: Inadequate blocking allows antibodies to bind non-specifically to the sample.

  • Solution: Optimize blocking by testing different blocking agents (BSA, normal serum, commercial blockers) and extending blocking time.

• Cross-reactivity with similar epitopes:

  • Problem: The antibody recognizes proteins with similar epitopes to SPCC1739.08c.

  • Solution: Perform pre-adsorption with related proteins or validate specificity using knockout controls.

• Fc receptor binding:

  • Problem: Fc portions of antibodies bind to Fc receptors in the sample.

  • Solution: Include Fc receptor blockers or use F(ab')2 fragments. Consider Fc modifications like N297A that reduce Fc receptor binding, similar to approaches used for therapeutic antibodies .

• High antibody concentration:

  • Problem: Excessive antibody increases non-specific interactions.

  • Solution: Perform careful titration experiments to determine the minimal effective concentration.

• Sample preparation issues:

  • Problem: Improper fixation can expose hydrophobic regions leading to non-specific binding.

  • Solution: Optimize fixation protocols and include detergents in wash buffers.

How can I troubleshoot weak or absent signal when using SPCC1739.08c antibody?

When facing weak or absent signals with SPCC1739.08c antibody, systematically investigate these potential causes:

• Epitope masking or destruction:

  • Problem: Fixation or sample preparation methods may alter the epitope.

  • Solution: Test different fixation methods (paraformaldehyde, methanol, acetone) and antigen retrieval techniques.

• Low target protein expression:

  • Problem: SPCC1739.08c may be expressed at low levels under your experimental conditions.

  • Solution: Consider signal amplification methods (tyramide signal amplification, polymer detection systems) or more sensitive detection methods.

• Antibody degradation:

  • Problem: Improper storage can lead to antibody degradation.

  • Solution: Store antibodies according to recommendations, avoiding freeze-thaw cycles, and aliquoting for long-term storage as advised for similar antibodies .

• Incorrect application parameters:

  • Problem: Suboptimal incubation times, temperatures, or buffer compositions.

  • Solution: Systematically optimize protocol parameters, testing different conditions in parallel.

• Detection system issues:

  • Problem: Secondary antibody or detection reagent problems.

  • Solution: Verify secondary antibody functionality with a positive control primary antibody, and check detection reagents with a control system.

How can I accurately quantify and statistically analyze immunofluorescence data from SPCC1739.08c antibody staining?

Rigorous quantification and statistical analysis of SPCC1739.08c immunofluorescence data requires:

These approaches ensure robust, reproducible quantification of immunofluorescence data, similar to strategies used in rigorous antibody validation studies .

How might SPCC1739.08c antibody be adapted for super-resolution microscopy applications?

Adapting SPCC1739.08c antibody for super-resolution microscopy requires specific considerations:

• Conjugation strategies for super-resolution compatible fluorophores:

  • Direct conjugation to photoswitchable fluorophores (Alexa Fluor 647, Atto 488)

  • Site-specific labeling using click chemistry to ensure optimal fluorophore positioning

  • Smaller detection probes like nanobodies or aptamers derived from the original antibody

• Optimization for specific super-resolution techniques:

  • STORM/PALM: Ensure proper photoswitching behavior in appropriate buffers

  • STED: Select fluorophores with appropriate depletion characteristics

  • SIM: Focus on signal strength and photostability

  • Expansion microscopy: Validate epitope preservation during expansion

• Validation approaches:

  • Compare with conventional microscopy to confirm specificity is maintained

  • Use correlative light and electron microscopy as a gold standard

  • Implement dual-color approaches with known interaction partners

• Data analysis considerations:

  • Implement clustering algorithms to analyze nanoscale distribution

  • Develop quantitative measures of spatial organization

  • Establish rigorous statistical frameworks for comparative analysis

These approaches build on established protocols for antibody optimization while addressing the unique requirements of super-resolution imaging techniques .

What are emerging applications for combining SPCC1739.08c antibody with genetic labeling approaches?

Innovative combinations of SPCC1739.08c antibody with genetic labeling create powerful research tools:

• Proximity labeling techniques:

  • BioID or TurboID fusion proteins to identify proteins in proximity to SPCC1739.08c

  • APEX2 labeling for electron microscopy correlation

  • Verification of proximity labeling results using co-immunoprecipitation with SPCC1739.08c antibody

• Genome editing for antibody validation:

  • CRISPR-Cas9 epitope tagging of endogenous SPCC1739.08c

  • Knock-in of fluorescent proteins for live-cell correlation

  • Creation of defined mutations to map the epitope recognized by the antibody

• Split protein complementation:

  • Combining antibody detection with split GFP or luciferase systems

  • Verification of protein-protein interactions identified through genetic screens

  • Development of hybrid detection systems for enhanced sensitivity

• Single-cell analysis integration:

  • Correlation of antibody staining with single-cell transcriptomics

  • Spatial transcriptomics combined with protein localization

  • Computational integration of protein and transcript datasets

These emerging approaches represent the frontier of cell biology research, combining the specificity of antibody detection with the precision of genetic manipulation .

How can computational approaches enhance the utility of SPCC1739.08c antibody in research?

Advanced computational methods significantly expand the research applications of SPCC1739.08c antibody:

• Machine learning for image analysis:

  • Automated segmentation of subcellular compartments

  • Classification of phenotypes following genetic or chemical perturbations

  • Extraction of subtle patterns undetectable by conventional analysis

• Systems biology integration:

  • Incorporation of antibody-derived localization data into protein interaction networks

  • Correlation with transcriptomic and proteomic datasets

  • Prediction of protein function based on localization and interaction patterns

• Virtual screening and in silico epitope prediction:

  • Computational modeling of antibody-antigen interactions

  • Prediction of cross-reactivity with related proteins

  • Design of improved antibodies through in silico affinity maturation

• Quantitative image analysis frameworks:

  • Development of standardized analysis pipelines for reproducibility

  • Integration of multiple imaging modalities

  • Spatial statistics for analyzing protein distribution patterns