SPAC222.17 Antibody

What is SPAC222.17 and what is its significance in fission yeast research?

SPAC222.17 is a protein in Schizosaccharomyces pombe (fission yeast) identified by the UniProt Number C6Y4B9. While detailed functional characterization appears limited in current literature, related proteins in the same genomic region (such as SPAC222.19) have been investigated in the context of complex formation with other proteins. Research suggests that proteins in this family may be involved in cellular regulatory pathways similar to the Ragulator complex, which plays roles in cellular growth regulation through TOR complex signaling . The antibody against SPAC222.17 provides a valuable tool for investigating this protein's expression, localization, and potential interactions in S. pombe.

What applications has the SPAC222.17 antibody been validated for?

The SPAC222.17 antibody has been validated for two primary applications:

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Western Blotting (WB)

These applications make the antibody suitable for both quantitative analysis of SPAC222.17 levels and qualitative detection of the protein in cellular extracts, allowing researchers to investigate expression patterns and post-translational modifications.

What components are provided with the SPAC222.17 antibody, and how should they be utilized?

The SPAC222.17 antibody package typically includes:

• 200μg antigens (intended as positive control)

• 1ml pre-immune serum (intended as negative control)

• Rabbit polyclonal antibodies purified by Antigen Affinity

This comprehensive set of components enables proper experimental design with appropriate controls. The positive control antigen helps verify antibody performance and establishes detection parameters, while the pre-immune serum serves as a critical negative control to identify potential non-specific binding. These controls should be incorporated into each experimental run to ensure reliable and interpretable results.

What is the recommended protocol for Western blot analysis using SPAC222.17 antibody?

For optimal Western blot results with SPAC222.17 antibody, we recommend following this protocol adapted from established S. pombe research methodologies:

• Sample Preparation:

  • Harvest cells in 10% trichloroacetic acid (TCA)

  • Prepare crude cell lysates according to established protocols

  • Determine protein concentration using Bio-Rad protein assay kit

• SDS-PAGE and Transfer:

  • Resolve proteins by SDS-PAGE

  • Transfer to nitrocellulose membrane

• Immunoblotting:

  • Block membrane with 5% non-fat milk in TBST

  • Incubate with SPAC222.17 antibody (recommended dilution: 1:1000)

  • Wash with TBST buffer (3 × 10 minutes)

  • Incubate with appropriate secondary antibody

  • Wash thoroughly with TBST

• Detection:

  • Develop using enhanced chemiluminescence (ECL) reagents

  • Exposure times may need optimization based on expression levels

For loading controls in S. pombe studies, anti-Spc1 antibody is commonly used as it provides consistent signals across various experimental conditions .

What immunoprecipitation protocol works best with SPAC222.17 antibody?

Based on protocols established for working with similar antibodies in S. pombe:

• Cell Preparation:

  • Grow cells to exponential phase in YES medium

  • Filter onto 0.45 μm mixed cellulose ester membrane

• Cell Lysis:

  • Disrupt cells in lysis buffer containing:

    • 20 mM HEPES-KOH (pH 7.5)

    • 150 mM potassium glutamate

    • 10% glycerol

    • 0.25% Tween-20

    • 10 mM sodium fluoride

    • 10 mM p-nitrophenylphosphate

    • 10 mM sodium pyrophosphate

    • 10 mM β-glycerophosphate

    • 0.1 mM sodium orthovanadate

    • Protease inhibitors (PMSF, leupeptin, and protease inhibitor cocktail)

  • Use glass beads with a Multi-beads Shocker or similar device

• Immunoprecipitation:

  • Clear lysate by centrifugation

  • Incubate with SPAC222.17 antibody or anti-FLAG/anti-myc beads for tagged versions

  • Collect immunocomplexes with Protein A/G beads

  • Wash extensively with lysis buffer

  • Elute proteins with SDS sample buffer or specific peptides for further analysis

For validation of novel interactions, serial immunoprecipitation with differently tagged proteins is recommended, followed by mass spectrometry analysis to identify complex components.

How can researchers optimize chromatin immunoprecipitation (ChIP) using SPAC222.17 antibody?

ChIP optimization for SPAC222.17 antibody should follow these key steps:

• Crosslinking Optimization:

  • Test different formaldehyde concentrations (0.5-1.5%)

  • Optimize crosslinking times (5-20 minutes) at room temperature

• Sonication Parameters:

  • Adjust sonication conditions to produce DNA fragments of 200-500 bp

  • Verify fragmentation efficiency by agarose gel electrophoresis

• Antibody Validation:

  • Perform titration experiments to determine optimal antibody concentration

  • Include IgG controls and input samples

• Immunoprecipitation Conditions:

  • Test different bead types (Protein A, Protein G, or magnetic beads)

  • Optimize incubation times and temperatures

• Washing Stringency:

  • Evaluate different wash buffer compositions to minimize background

  • Consider including a pre-clearing step to reduce non-specific binding

• Analysis Methods:

  • Use qPCR for targeted analysis or sequencing for genome-wide assessments

  • Compare ChIP profiles with those obtained using standard antibodies (e.g., anti-Pol II) to ensure comparable signal distribution

Research indicates that ChIP profiles obtained with different antibodies targeting the same complex often show >95% peak overlap when optimized correctly .

What approaches can researchers use to characterize potential protein complexes involving SPAC222.17?

To characterize protein complexes involving SPAC222.17, consider these approaches:

• Tandem Affinity Purification:

  • Generate strains with tagged SPAC222.17 (e.g., FLAG, myc)

  • Perform successive immunoprecipitation procedures

  • Analyze co-purified proteins by mass spectrometry

• Co-immunoprecipitation Experiments:

  • Use SPAC222.17 antibody for immunoprecipitation

  • Identify interaction partners through immunoblotting or mass spectrometry

  • Validate interactions through reciprocal co-immunoprecipitation

• Two-hybrid Analysis:

  • Screen for interaction partners using SPAC222.17 as bait

  • Confirm positive interactions by subsequent biochemical assays

• Fluorescence Microscopy:

  • Visualize protein co-localization using fluorescently tagged proteins

  • Analyze cellular distribution patterns in different growth conditions

  • Collect Z-axial images and perform deconvolution for accurate spatial analysis

This strategy has successfully identified novel protein complexes in S. pombe, as demonstrated with the Lam protein complex formation, where similar approaches revealed interactions between previously uncharacterized proteins .

What are common issues when using SPAC222.17 antibody and how can they be resolved?

How should the SPAC222.17 antibody be stored and handled for optimal performance?

To maintain optimal performance of the SPAC222.17 antibody:

• Storage Conditions:

  • Store antibody at -20°C or -80°C for long-term storage

  • Avoid repeated freeze-thaw cycles by preparing single-use aliquots

• Working Solution Preparation:

  • Thaw aliquots on ice

  • Centrifuge briefly before opening to collect solution at the bottom

  • Prepare working dilutions fresh on the day of use

• Stability Considerations:

  • Working dilutions may be stored at 4°C for up to one week

  • Monitor antibody performance periodically with positive controls

  • Document lot numbers and performance to track potential variability

• Contamination Prevention:

  • Use sterile techniques when handling the antibody

  • Avoid contamination with microorganisms or chemicals

  • Include sodium azide (0.02%) for longer-term storage of working dilutions

What controls should be included when using SPAC222.17 antibody in immunological assays?

A comprehensive experimental design with SPAC222.17 antibody should include:

• Positive Controls:

  • Recombinant SPAC222.17 protein (200μg supplied with antibody)

  • Lysates from wild-type S. pombe expressing normal levels of SPAC222.17

  • Over-expression samples if available

• Negative Controls:

  • Pre-immune serum (1ml supplied with antibody)

  • Lysates from SPAC222.17 deletion strains (if viable)

  • IgG control matched to host species (rabbit)

• Loading/Normalization Controls:

  • Anti-Spc1 antibody for protein normalization in S. pombe studies

  • Total protein staining (Ponceau S, SYPRO Ruby) for transfer verification

• Procedural Controls:

  • Secondary antibody only control to identify non-specific binding

  • Peptide competition assay to demonstrate specificity

  • Cross-reactivity assessment with related proteins

• Validation Approach:

  • Use of multiple detection techniques (Western blot, immunofluorescence)

  • Correlation of results with orthogonal methods (RT-PCR, RNA-seq)

How can researchers verify the specificity of SPAC222.17 antibody?

To verify antibody specificity, researchers should implement these approaches:

• Genetic Validation:

  • Compare signal in wild-type vs. SPAC222.17 knockout/knockdown strains

  • Test for signal reduction in strains with reduced expression

• Molecular Validation:

  • Perform peptide competition assays using the immunizing antigen

  • Assess cross-reactivity with related proteins by recombinant protein analysis

• Technical Validation:

  • Compare results from multiple lots of the antibody

  • Use alternative antibodies targeting different epitopes of SPAC222.17

  • Validate findings using tagged versions of the protein

• Functional Validation:

  • Correlate antibody detection with expected localization patterns

  • Confirm antibody detects changes in expression under conditions known to affect the protein

  • Verify detection of protein-protein interactions through co-immunoprecipitation

How can SPAC222.17 antibody be used in combination with mass spectrometry for protein complex analysis?

Integration of immunoprecipitation with mass spectrometry requires:

• Sample Preparation Workflow:

  • Perform immunoprecipitation using SPAC222.17 antibody

  • Resolve samples by SDS-PAGE

  • Slice gel into multiple pieces for in-gel trypsin digestion

• Mass Spectrometry Analysis:

  • Analyze digested samples using LTQ-Orbitrap or similar MS system

  • Compare MS/MS spectra against protein databases (e.g., NCBInr) with taxonomy restricted to S. pombe

  • Use appropriate search engines (e.g., Mascot server) for peptide identification

• Data Analysis Considerations:

  • Filter results based on coverage, number of unique peptides, and score

  • Compare with control immunoprecipitations to identify specific interactors

  • Validate key interactions through targeted approaches (Western blot, co-IP)

• Quantitative Analysis:

  • Consider SILAC or TMT labeling for quantitative comparison across conditions

  • Analyze changes in interaction partners under different cellular states

  • Correlate complex formation with functional outcomes

This approach has successfully identified novel protein complexes in S. pombe, providing insights into cellular pathways and regulatory mechanisms .

What fluorescence microscopy approaches work best for cellular localization studies using SPAC222.17 antibody?

For optimal fluorescence microscopy studies:

• Sample Preparation:

  • Grow cells exponentially in EMM liquid medium

  • Fix cells with appropriate fixation method (e.g., formaldehyde)

  • Perform membrane permeabilization and blocking

• Immunofluorescence Protocol:

  • Incubate with SPAC222.17 antibody at optimized dilution

  • Use appropriate fluorescent secondary antibodies

  • Include nuclear and vacuolar staining for spatial reference

• Imaging Parameters:

  • Capture Z-axial images at 0.4 μm intervals using 100X objective lens

  • Perform deconvolution using appropriate software (e.g., DeltaVision SoftWoRx)

  • Analyze >200 cells per condition for statistical significance

• Co-localization Studies:

  • Combine with markers for specific cellular compartments

  • Use FM4-64 dye for vacuole visualization

  • Consider tagged versions of SPAC222.17 (GFP, mCherry) for live-cell imaging

• Advanced Imaging Techniques:

  • Consider super-resolution microscopy for detailed localization

  • Use FRAP or photoactivation to assess protein dynamics

  • Implement time-lapse imaging to monitor changes during cell cycle progression

These approaches allow comprehensive assessment of protein localization and dynamics in response to various cellular conditions and genetic backgrounds.

How might SPAC222.17 antibody contribute to understanding protein function in relation to cellular pathways?

The SPAC222.17 antibody could facilitate several innovative research directions:

• Pathway Integration Analysis:

  • Investigation of SPAC222.17's potential role in TOR signaling pathways

  • Comparison with related proteins involved in Ragulator and GATOR1 complexes

  • Assessment of interactions with GTPases and their regulatory factors

• Evolutionary Conservation Studies:

  • Comparative analysis across yeast species (S. pombe, S. cerevisiae)

  • Identification of functional homologs in higher eukaryotes

  • Exploration of structural similarities despite limited sequence homology

• Stress Response Characterization:

  • Analysis of SPAC222.17 expression and localization under various stress conditions

  • Investigation of post-translational modifications in response to cellular signals

  • Correlation with changes in cellular growth and division

• Cell Cycle Regulation:

  • Assessment of protein levels and modifications throughout the cell cycle

  • Investigation of potential roles in meiotic processes

  • Correlation with known cell cycle regulators

These approaches could reveal novel insights into fundamental cellular processes and regulatory mechanisms conserved across eukaryotes.

What are the key considerations for researchers new to working with SPAC222.17 antibody?

Researchers beginning work with SPAC222.17 antibody should consider:

• Experimental Planning:

  • Start with validated applications (ELISA, Western blot)

  • Include all appropriate controls (both positive and negative)

  • Optimize antibody dilutions for each application

• Technical Approach:

  • Store antibody according to manufacturer recommendations (-20°C or -80°C)

  • Validate antibody performance in your specific experimental system

  • Document detailed protocols for reproducibility

• Data Interpretation:

  • Consider potential cross-reactivity with related proteins

  • Correlate antibody-based findings with orthogonal techniques

  • Interpret results in the context of known S. pombe biology

• Methodology Development:

  • Begin with established protocols for S. pombe protein analysis

  • Systematically optimize each step for SPAC222.17 detection

  • Create standardized protocols specific to your research questions