SPCC594.04c Antibody

What is SPCC594.04c protein and why is it studied in research?

SPCC594.04c is an uncharacterized protein from Schizosaccharomyces pombe (fission yeast) that belongs to the steroid oxidoreductase superfamily (predicted) . This protein is of interest to researchers studying fundamental cellular processes in eukaryotic model organisms. S. pombe serves as an excellent model system for investigating conserved cellular mechanisms due to its similarity to higher eukaryotes in cell cycle regulation, chromosome structure, and RNA processing pathways . The antibodies against this protein enable detection and study of its expression, localization, and potential functions in cellular processes.

What applications are suitable for SPCC594.04c antibodies in S. pombe research?

SPCC594.04c antibodies are primarily used in Western Blot (WB) and ELISA applications to identify and quantify the protein . These antibodies are generated in rabbits against S. pombe (strain 972/24843) and purified through antigen-affinity methods, resulting in IgG-type antibodies with high specificity . When conducting research with these antibodies, it's essential to include appropriate controls and validate the antibody using known positive samples to confirm specificity, especially since SPCC594.04c is an uncharacterized protein.

What is the difference between using recombinant SPCC594.04c protein and antibodies against it?

The choice depends on your experimental goals:

• Recombinant SPCC594.04c protein (available from E. coli, yeast, baculovirus, or mammalian cell expression systems ) is typically used as:

  • A positive control in Western blot or immunoprecipitation experiments

  • An immunogen for generating custom antibodies

  • A substrate in enzymatic assays to characterize protein function

  • A protein standard for quantification purposes

• SPCC594.04c antibodies are used to:

  • Detect endogenous SPCC594.04c protein in cell or tissue samples

  • Study protein expression levels under different conditions

  • Localize the protein within cellular compartments using immunofluorescence

  • Immunoprecipitate the protein for protein-protein interaction studies

Both tools complement each other in a comprehensive research approach to uncharacterized proteins .

How should I optimize Western blot conditions when using SPCC594.04c antibody?

When optimizing Western blot conditions for SPCC594.04c antibody, consider the following methodological approach:

• Sample preparation: Fission yeast cells should be lysed using glass bead disruption in a buffer containing protease inhibitors to prevent degradation.

• Protein denaturation: Test both reducing and non-reducing conditions, as the steroid oxidoreductase superfamily proteins may have important disulfide bonds.

• Antibody dilution series: Begin with a 1:1000 dilution and perform a titration (1:500, 1:1000, 1:2000, 1:5000) to determine optimal signal-to-noise ratio.

• Blocking optimization: Compare 5% non-fat milk versus 3-5% BSA in TBST as blocking agents.

• Incubation conditions: Test both overnight incubation at 4°C and 2-hour incubation at room temperature.

• Controls: Include both positive controls (purified recombinant SPCC594.04c protein) and negative controls (lysate from a SPCC594.04c deletion strain if available).

• Detection system: HRP-conjugated secondary antibodies with chemiluminescent detection are recommended for high sensitivity .

The antibody has demonstrated ≥85% purity as determined by SDS-PAGE , making it suitable for most standard immunodetection methods with proper optimization.

What are the recommended immunoprecipitation protocols for SPCC594.04c antibody?

For successful immunoprecipitation using SPCC594.04c antibody, follow this methodological workflow:

• Cell lysis: Lyse S. pombe cells in a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, and protease inhibitor cocktail.

• Pre-clearing: Incubate lysate with Protein G beads for 1 hour at 4°C to reduce non-specific binding.

• Antibody binding: Add 2-5 μg of SPCC594.04c antibody to 500 μg of pre-cleared lysate and incubate overnight at 4°C with gentle rotation.

• Immunoprecipitation: Add 30 μl of Protein G beads and incubate for 2-4 hours at 4°C.

• Washing: Wash beads 4-5 times with lysis buffer containing reduced detergent concentration.

• Elution: Elute bound proteins by boiling in SDS sample buffer.

• Analysis: Analyze by Western blot using the same SPCC594.04c antibody or antibodies against potential interacting partners.

This approach has been successfully used to identify protein-protein interactions in S. pombe, particularly for components involved in RNA polymerase II transcription regulation pathways .

How can I use SPCC594.04c antibody in chromatin immunoprecipitation (ChIP) experiments?

While SPCC594.04c has not been specifically documented in ChIP experiments, the protocol can be adapted based on successful ChIP studies in S. pombe:

• Crosslinking: Treat S. pombe cells with 1% formaldehyde for 15 minutes at room temperature.

• Chromatin preparation: Lyse cells and sonicate to generate DNA fragments of 200-500 bp.

• Immunoprecipitation:

  • Pre-clear chromatin with Protein G beads

  • Incubate 5-10 μg of SPCC594.04c antibody with chromatin overnight at 4°C

  • Add Protein G beads and incubate for 2-3 hours at 4°C

  • Wash with increasing stringency buffers

• Reversal of crosslinks: Reverse crosslinks at 65°C overnight.

• DNA purification: Purify DNA using phenol-chloroform extraction or commercial kits.

• Analysis: Analyze by qPCR or next-generation sequencing.

For genome-wide binding profiles, consider adapting the ChIP-chip methodology used for studying transcription factors in S. pombe, where crosslinked chromatin is immunoprecipitated and then hybridized to tiling arrays or sequenced .

How can I determine if SPCC594.04c is part of a protein complex using its antibody?

To investigate protein complex formation involving SPCC594.04c, consider this comprehensive approach:

• Co-immunoprecipitation followed by mass spectrometry:

  • Perform immunoprecipitation using SPCC594.04c antibody

  • Analyze the precipitated proteins by LC-MS/MS

  • Validate potential interactions with reciprocal co-IPs

• Size exclusion chromatography:

  • Fractionate S. pombe cell extracts by gel filtration

  • Analyze fractions by Western blot using SPCC594.04c antibody

  • Compare elution profile with known molecular weight standards and other proteins of interest

• Blue native PAGE:

  • Separate native protein complexes using blue native PAGE

  • Perform Western blotting with SPCC594.04c antibody

  • Identify potential complex sizes

• Proximity-dependent biotin identification (BioID):

  • Create a fusion of SPCC594.04c with a biotin ligase

  • Identify biotinylated proteins in proximity using streptavidin pulldown and mass spectrometry

  • Validate interactions using the SPCC594.04c antibody

This multi-method approach has proven effective for characterizing novel protein complexes in S. pombe, particularly for transcription-related proteins .

How can I use SPCC594.04c antibody to investigate its potential role in transcription?

Based on studies of RNA polymerase II transcription in S. pombe, consider the following research strategy:

• ChIP-seq analysis:

  • Perform chromatin immunoprecipitation with SPCC594.04c antibody

  • Sequence precipitated DNA to identify genomic binding sites

  • Analyze binding patterns relative to transcription start sites, gene bodies, and termination sites

• RNA-seq in deletion strains:

  • Generate a SPCC594.04c deletion strain

  • Compare gene expression profiles with wild-type using RNA-seq

  • Identify genes with altered expression patterns

• Co-localization with RNA polymerase II:

  • Perform sequential ChIP (ChIP-reChIP) with SPCC594.04c antibody and RNA polymerase II antibody

  • Determine if they co-occupy the same genomic regions

• Nascent transcription assays:

  • Use nuclear run-on or BrUTP incorporation assays

  • Combine with SPCC594.04c immunoprecipitation to assess association with actively transcribing complexes

This approach has been successfully employed to characterize the role of other S. pombe proteins in transcriptional regulation .

How can I validate SPCC594.04c antibody specificity in my experimental system?

Rigorous validation of antibody specificity is critical for reliable research outcomes:

• Genetic controls:

  • Test the antibody on samples from SPCC594.04c deletion strain (negative control)

  • Test on samples with epitope-tagged SPCC594.04c (positive control)

  • Compare signal intensity with wild-type samples

• Peptide competition assay:

  • Pre-incubate the antibody with excess purified SPCC594.04c protein or immunizing peptide

  • Perform Western blot on wild-type samples with both blocked and unblocked antibody

  • Specific signals should be significantly reduced with the blocked antibody

• Cross-reactivity assessment:

  • Test the antibody against recombinant SPCC594.04c protein expressed in different systems

  • Test against closely related proteins if identified through sequence analysis

  • Perform immunoprecipitation followed by mass spectrometry to identify all bound proteins

• Correlation of protein levels with transcript levels:

  • Manipulate SPCC594.04c transcript levels (overexpression or RNAi)

  • Confirm changes in transcript levels by RT-qPCR

  • Verify corresponding changes in protein levels detected by the antibody

These validation steps are essential for ensuring the reliability of subsequent experimental results, especially for uncharacterized proteins like SPCC594.04c .

What are the common causes of non-specific binding when using SPCC594.04c antibody?

When encountering non-specific binding with SPCC594.04c antibody, consider these potential causes and solutions:

• High antibody concentration:

  • Problem: Excessive antibody can bind non-specifically

  • Solution: Perform a titration series to determine optimal concentration (typically 1:1000-1:5000 for Western blot)

• Insufficient blocking:

  • Problem: Incomplete blocking leads to non-specific binding

  • Solution: Increase blocking time (1-2 hours) or concentration (5% BSA or milk) and ensure complete coverage

• Cross-reactivity with related proteins:

  • Problem: The antibody may recognize epitopes shared with related proteins

  • Solution: Use more stringent washing conditions and potentially pre-absorb the antibody with recombinant related proteins

• Sample preparation issues:

  • Problem: Inadequate lysis or denaturation can expose epitopes non-specifically

  • Solution: Optimize lysis conditions and ensure complete protein denaturation for Western blots

• Post-translational modifications:

  • Problem: PTMs may alter antibody recognition

  • Solution: Test different sample preparation methods that preserve or remove specific modifications

Proper controls, including a SPCC594.04c deletion strain and recombinant protein controls, are essential for distinguishing specific from non-specific signals .

How can I resolve contradictory results between antibody-based detection and genomic/transcriptomic data?

When faced with discrepancies between antibody-based SPCC594.04c detection and genomic/transcriptomic data, investigate using this systematic approach:

• Verify transcript expression:

  • Confirm SPCC594.04c mRNA expression using RT-qPCR with multiple primer sets

  • Compare with RNA-seq data, focusing on exon coverage patterns

  • Check for alternative splicing or transcript isoforms

• Assess protein stability and turnover:

  • Perform cycloheximide chase experiments to assess protein half-life

  • Test if proteasome inhibitors affect detected protein levels

  • Consider whether environmental conditions affect protein stability

• Evaluate post-transcriptional regulation:

  • Investigate potential miRNA regulation of SPCC594.04c

  • Assess translational efficiency through polysome profiling

  • Check for RNA-binding protein interactions that might affect translation

• Technical factors:

  • Confirm antibody specificity using the methods in FAQ 4.3

  • Test multiple antibody lots if available

  • Consider epitope accessibility issues in different experimental contexts

• Biological context:

  • Test different growth conditions and cell cycle stages

  • Examine strain-specific variations

  • Consider whether SPCC594.04c undergoes regulated degradation

This systematic evaluation can help resolve apparent contradictions and may reveal interesting regulatory mechanisms governing SPCC594.04c expression .

How should I interpret SPCC594.04c localization data from immunofluorescence compared to fractionation studies?

When analyzing potentially conflicting localization data, consider these methodological aspects:

• Technical differences:

  • Immunofluorescence shows proteins in situ but may have accessibility limitations

  • Fractionation biochemically separates compartments but may introduce artifacts during extraction

  • Compare fixation methods (formaldehyde vs. methanol) which differentially preserve structures

• Resolution limitations:

  • Standard immunofluorescence may not resolve fine structures

  • Consider super-resolution microscopy (STED, PALM, STORM) for more detailed localization

  • Correlate with electron microscopy using immunogold labeling for highest resolution

• Dynamic localization:

  • Perform time-course experiments under different conditions

  • Use live-cell imaging with fluorescently tagged SPCC594.04c to track movement

  • Compare with known markers of subcellular compartments

• Quantitative assessment:

  • Quantify signal distribution across cellular compartments

  • Determine relative enrichment in different fractions

  • Use statistical analysis to evaluate significance of localization patterns

• Functional validation:

  • Create targeted mutations that disrupt putative localization signals

  • Perform functional assays in different compartments

  • Correlate localization with interaction partners

This comprehensive approach can resolve apparent discrepancies and provide insight into the dynamic nature of SPCC594.04c localization and function .

How can I apply computational antibody design methods to improve SPCC594.04c antibody specificity?

Based on recent advances in computational antibody design, researchers can improve SPCC594.04c antibody specificity through:

• Epitope refinement using structural prediction:

  • Generate structural models of SPCC594.04c using AlphaFold2

  • Identify unique, surface-exposed epitopes that distinguish it from related proteins

  • Design synthetic peptides targeting these unique regions for antibody production

• In silico antibody optimization:

  • Apply RosettaAntibodyDesign (RAbD) framework to model antibody-antigen interactions

  • Modify CDR sequences to enhance binding affinity and specificity

  • Validate improved designs through in vitro binding assays

• Machine learning approaches:

  • Train algorithms on existing antibody-epitope data to predict optimal binding regions

  • Identify potential cross-reactive epitopes to avoid

  • Design multi-epitope recognition strategies for increased specificity

• Molecular dynamics simulations:

  • Simulate antibody-antigen binding events to identify stable interaction interfaces

  • Optimize binding energy through targeted mutations

  • Predict effects of different buffer conditions on binding specificity

This computational approach, combined with experimental validation, can significantly improve antibody specificity and performance in challenging applications .

How can the SPCC594.04c antibody be used in studying potential moonlighting functions of this protein?

To investigate potential moonlighting functions of SPCC594.04c:

• Differential interactome analysis:

  • Perform immunoprecipitation with SPCC594.04c antibody under different cellular conditions

  • Analyze interacting partners by mass spectrometry

  • Look for condition-specific interactions that suggest context-dependent functions

• Subcellular redistribution studies:

  • Track localization of SPCC594.04c using the antibody under various stresses

  • Correlate relocalization with acquisition of alternate functions

  • Use proximity labeling (BioID/APEX) to identify neighborhood proteins in each location

• Post-translational modification mapping:

  • Immunoprecipitate SPCC594.04c and analyze PTMs by mass spectrometry

  • Correlate specific modifications with different functional states

  • Generate modification-specific antibodies for functional studies

• Comparative analysis across species:

  • Use the antibody (if cross-reactive) or generate antibodies against orthologs

  • Compare localization and interaction patterns across evolutionary distance

  • Identify conserved versus divergent functions

This multifaceted approach can uncover unexpected roles of SPCC594.04c beyond its predicted function as a steroid oxidoreductase superfamily protein .

How can I integrate SPCC594.04c antibody-based studies with CRISPR-Cas9 genome editing for functional characterization?

Combining antibody-based detection with CRISPR-Cas9 technology enables powerful functional studies:

• Endogenous tagging strategies:

  • Use CRISPR to introduce epitope tags at the endogenous SPCC594.04c locus

  • Compare detection using both epitope antibodies and SPCC594.04c-specific antibody

  • Validate antibody specificity against the tagged strain

• Domain-specific functional analysis:

  • Generate CRISPR-mediated domain deletions or point mutations

  • Use the antibody to assess expression, stability, and localization of mutant proteins

  • Correlate structural changes with functional outcomes

• Conditional degradation systems:

  • Implement auxin-inducible or temperature-sensitive degron tags via CRISPR

  • Monitor protein depletion kinetics using the antibody

  • Correlate protein levels with phenotypic changes

• CRISPRi transcriptional control:

  • Establish dCas9-based transcriptional regulation of SPCC594.04c

  • Use the antibody to precisely measure resulting protein level changes

  • Create a titratable system to determine threshold levels for different functions

• Synthetic genetic interaction screens:

  • Combine CRISPR-mediated gene deletions with antibody-based detection

  • Screen for genetic interactions that affect SPCC594.04c expression or localization

  • Identify regulatory networks and functional pathways

This integrated approach maximizes the utility of both CRISPR technology and antibody-based detection for comprehensive functional characterization .