SPAC17G8.12 Antibody

What is SPAC17G8.12 protein and what is its significance in research?

SPAC17G8.12 is an uncharacterized protein in Schizosaccharomyces pombe (fission yeast) with UniProt accession number Q10324. Based on genomic context analysis, it appears to be associated with transcriptional regulation processes in S. pombe . The significance of this protein relates to understanding fundamental transcriptional mechanisms in eukaryotic systems, as it has been categorized alongside other transcription-related proteins like med20, which is a Mediator complex subunit .

Methodologically, researchers investigating this protein should consider:

• Comparative genomic approaches to identify potential functional domains

• Gene expression analysis under various cellular conditions

• Protein-protein interaction studies to determine binding partners

• Functional studies using knockout or knockdown methodologies

What are the optimal experimental conditions for using SPAC17G8.12 antibody?

For optimal experimental results with SPAC17G8.12 antibody:

Buffer System:

• The antibody is supplied in a buffer containing 0.03% Proclin 300 as a preservative, 50% Glycerol, and 0.01M PBS at pH 7.4

• For experimental applications, maintain similar buffer conditions during dilution to preserve antibody activity

Working Concentrations:

• For Western blotting: Start with 1:500-1:2000 dilution and optimize based on signal strength

• For immunoprecipitation: 2-5 µg per reaction is typically appropriate

• For immunofluorescence: Begin with 1:100-1:500 dilution

Control experiments should include:

• A negative control lacking primary antibody

• A sample from knockout/knockdown S. pombe strains if available

• A positive control with overexpressed SPAC17G8.12 protein

How should samples be prepared for optimal SPAC17G8.12 detection?

Sample preparation methodology significantly impacts detection quality:

For cell lysate preparation:

• Grow S. pombe cells to mid-log phase in appropriate media

• Harvest cells by centrifugation (3,000 × g for 5 minutes)

• Wash cell pellet twice with ice-cold PBS

• Lyse cells using either:

  • Mechanical disruption with glass beads in lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, with protease inhibitors)

  • Enzymatic digestion with lysing enzymes followed by detergent treatment

For protein extraction from subcellular fractions:

• Perform differential centrifugation to isolate nuclear fractions

• Extract nuclear proteins using high-salt buffer (420 mM NaCl, 20 mM HEPES pH 7.9, 1.5 mM MgCl₂, 0.2 mM EDTA, 25% glycerol)

• Quantify protein concentration using BCA or Bradford assay before loading onto gels

What validation methods should be employed to confirm SPAC17G8.12 antibody specificity?

Rigorous validation is essential for antibody-based experiments:

• Western Blot Validation:

  • Compare band pattern between wild-type and SPAC17G8.12 knockout strains

  • Perform peptide competition assay using the immunizing peptide

  • Test cross-reactivity against related S. pombe proteins

• Immunoprecipitation Validation:

  • Confirm pulled-down protein by mass spectrometry

  • Verify absence of signal in knockout controls

• Specificity Controls:

  • Test antibody against recombinantly expressed SPAC17G8.12 protein

  • Perform siRNA knockdown and observe corresponding reduction in signal

Similar validation approaches have been demonstrated for other S. pombe proteins, such as the methodology used for Sre1-specific antibody validation, which confirmed specificity by loss of immunoreactivity in an sre1Δ strain .

How does SPAC17G8.12 relate to transcriptional regulation networks in S. pombe?

Based on available research, SPAC17G8.12 appears in the transcription functional category alongside other important transcription-related proteins . The protein may be part of transcriptional regulation networks similar to:

Research approaches to elucidate its role should include:

• ChIP-seq analysis to identify genomic binding sites

• RNA-seq after knockout/knockdown to determine affected gene networks

• Co-immunoprecipitation studies with known transcription factors

• Protein domain analysis and comparison with related transcriptional regulators

What methodological approaches are most effective for studying SPAC17G8.12's potential role in oxygen-responsive pathways?

Given the association of nearby genes with oxygen-responsive regulation , researchers investigating SPAC17G8.12's potential role in oxygen response should consider:

Experimental Design:

• Compare gene expression and protein levels under normoxic versus hypoxic conditions

• Utilize an InVivo hypoxic work station similar to that used for Sre1 cleavage assays

• Implement time-course experiments to capture dynamic responses

• Combine with genetic approaches (knockout/overexpression)

Analytical Methods:

• Western blotting under reducing and non-reducing conditions

• RT-qPCR for transcriptional analysis

• Proteomic profiling using mass spectrometry

• Functional assays to detect changes in cellular physiology

Based on research with Mga2, which regulates oxygen-responsive lipid homeostasis in S. pombe , similar methodological approaches could reveal whether SPAC17G8.12 participates in parallel or intersecting pathways.

How can researchers analyze potential interactions between SPAC17G8.12 and chromatin remodeling complexes?

Given the proximity of SPAC17G8.12 to SPAC17G8.13c, which shows similarity to MYST family histone acetyltransferases , methodological approaches for investigating potential chromatin-related functions include:

• Chromatin Immunoprecipitation (ChIP):

  • Use SPAC17G8.12 antibody to pull down associated chromatin

  • Sequence associated DNA to identify genomic binding sites

  • Perform ChIP-qPCR for targeted analysis of specific genomic regions

• Co-Immunoprecipitation Studies:

  • Pull down SPAC17G8.12 and identify interacting proteins by mass spectrometry

  • Perform reciprocal IP with known chromatin modifiers

  • Test interactions with specific histone marks using appropriate antibodies

• Histone Modification Analysis:

  • Compare histone modification patterns in wild-type versus SPAC17G8.12 knockout strains

  • Utilize antibodies against specific histone marks (H3K9ac, H3K4me3, etc.)

  • Combine with transcriptional analysis to correlate changes

What are the considerations for multiplexing SPAC17G8.12 antibody with other antibodies in multi-parameter experiments?

For complex experimental designs involving multiple antibodies:

Technical Considerations:

• Antibody Compatibility:

  • Ensure host species compatibility to avoid cross-reactivity

  • Select antibodies with distinct fluorophores if using immunofluorescence

  • For Western blotting, consider antibodies that detect proteins of different molecular weights

• Optimization Protocol:

  • Test each antibody individually before multiplexing

  • Determine optimal blocking conditions (BSA vs. milk proteins)

  • Establish sequence of antibody addition (primary followed by secondary)

• Signal Separation:

  • For fluorescence-based detection, select fluorophores with minimal spectral overlap

  • In chemiluminescence, use sequential detection with stripping between antibodies

  • Consider specialized multiplexing systems like Odyssey CLx infrared imaging

How can researchers integrate SPAC17G8.12 studies with broader investigations of transcriptional networks in fission yeast?

Integrative Research Methodology:

• Network Analysis Approach:

  • Combine protein-protein interaction data with transcriptome profiling

  • Utilize gene ontology enrichment analysis for functional clustering

  • Apply computational modeling to predict regulatory relationships

• Comparative Genomics:

  • Analyze conservation patterns across yeast species

  • Identify functional orthologs in S. cerevisiae and other model organisms

  • Compare with known transcriptional regulators like Mga2

• Multi-omics Integration:

  • Correlate transcriptome, proteome, and chromatin structure data

  • Apply systems biology approaches to construct comprehensive regulatory networks

  • Use machine learning algorithms to identify patterns in complex datasets

• Genetic Interaction Mapping:

  • Perform synthetic genetic array analysis

  • Test for genetic interactions with known transcription factors

  • Create double mutants with genes involved in oxygen-responsive pathways

This integrative approach would position SPAC17G8.12 research within the broader context of transcriptional regulation in eukaryotic systems, potentially revealing important functional insights.

What are the common challenges in SPAC17G8.12 antibody applications and how can they be addressed?

Common technical challenges and their methodological solutions include:

• Weak Signal Detection:

  • Increase antibody concentration (reduce dilution factor)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use signal enhancement systems (biotin-streptavidin amplification)

  • Optimize protein extraction protocol to increase target protein yield

• High Background:

  • Increase blocking stringency (5% BSA instead of 3%)

  • Add 0.1-0.3% Tween-20 to washing buffers

  • Pre-absorb antibody with cell lysate from knockout strain

  • Use more stringent washing steps (increase number and duration)

• Inconsistent Results:

  • Standardize protein quantification methods (BCA protein assay)

  • Verify equal loading by Ponceau S staining after transfer

  • Utilize internal loading controls appropriate for S. pombe

  • Maintain consistent experimental conditions across replicates

What quantitative methods can be applied to SPAC17G8.12 antibody-based experiments?

For rigorous quantitative analysis:

• Densitometric Analysis Protocol:

  • Capture Western blot images using a linear detection system (e.g., Odyssey CLx)

  • Analyze band intensity using ImageJ or similar software

  • Normalize to appropriate loading controls

  • Apply statistical analysis across multiple biological replicates

• Quantitative Immunofluorescence:

  • Use confocal microscopy with consistent acquisition settings

  • Measure signal intensity within defined cellular compartments

  • Include calibration standards for absolute quantification

  • Apply appropriate statistical tests for comparative analysis

• ELISA-Based Quantification:

  • Develop a sandwich ELISA using SPAC17G8.12 antibody

  • Generate standard curves with recombinant protein

  • Implement rigorous statistical methods for data analysis

  • Validate results across multiple experimental runs