SPAC29A4.23 Antibody

What is known about the SPAC29A4.23 protein in Schizosaccharomyces pombe?

SPAC29A4.23 is a protein encoded in the Schizosaccharomyces pombe genome (strain 972 / ATCC 24843), with UniProt accession number P0CU25. Based on the nomenclature, this protein is located on chromosome I of the fission yeast genome. While the specific function of SPAC29A4.23 is not extensively characterized in the available literature, it likely plays a role in cellular processes typical of fission yeast proteins.

For functional characterization, researchers typically employ:

• Sequence homology analysis with related proteins

• Phenotypic assessment of deletion mutants

• Protein localization studies using tagged constructs

• Expression profiling under different conditions

• Interaction studies to identify binding partners

Understanding the biological context of SPAC29A4.23 is crucial for designing appropriate experimental controls and interpreting antibody-based detection results accurately.

What experimental applications is SPAC29A4.23 Antibody suitable for in fission yeast research?

SPAC29A4.23 Antibody can be employed across multiple experimental techniques commonly used in yeast research:

For each application, optimization of antibody concentration, incubation conditions, and detection methods will be necessary to achieve reliable results .

How can researchers optimize protocols for immunolocalization studies using SPAC29A4.23 Antibody?

Successful immunolocalization in S. pombe requires careful consideration of several methodological aspects:

Sample Preparation:

• Methanol fixation is particularly effective for preserving S. pombe cellular structures

• Cell wall digestion may be necessary for improved antibody penetration

• Spheroplasting protocols can enhance accessibility to intracellular antigens

Staining Protocol:

• Begin with 1:100 to 1:500 antibody dilutions for optimization

• Incubate primary antibody (SPAC29A4.23) for 2-16 hours at 4°C

• Use appropriate blocking solution (typically 1-5% BSA) to reduce background

• Include 0.1% Triton X-100 or similar detergent for permeabilization if needed

Visualization:

• Combine with DAPI staining (1 μg/ml) for nuclear visualization

• Consider co-staining with cell wall markers (e.g., Aniline blue for β-1,3-glucan)

• Use appropriate filters compatible with secondary antibody fluorophores

For quantitative analysis, maintain consistent exposure settings and acquisition parameters across experimental and control samples .

What are the best approaches for validating the specificity of SPAC29A4.23 Antibody in S. pombe?

Rigorous validation is essential to ensure experimental reliability:

Genetic Controls:

• Test antibody reactivity in wild-type versus SPAC29A4.23 deletion strains

• Compare with strains expressing tagged versions (e.g., SPAC29A4.23-GFP)

• Examine cross-reactivity with closely related proteins

Biochemical Validation:

• Peptide competition assays using the immunizing peptide

• Western blot analysis to confirm detection at the expected molecular weight

• Pre-absorption controls to identify non-specific binding

Advanced Validation Techniques:

• Multiple independent antibodies targeting different epitopes

• siRNA knockdown to confirm signal reduction correlates with protein depletion

• Mass spectrometry analysis of immunoprecipitated proteins

These validation approaches should be documented systematically, as they provide critical evidence for antibody specificity that reviewers often require in publications .

What troubleshooting strategies should be employed when using SPAC29A4.23 Antibody in Western blot applications?

When encountering issues with Western blot detection of SPAC29A4.23, consider these systematic troubleshooting approaches:

No Signal or Weak Signal:

• Optimize antibody concentration (test serial dilutions from 1:100 to 1:5000)

• Increase protein loading (20-50 μg of total protein)

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

• Use more sensitive detection methods (ECL Prime or SuperSignal West Femto)

• Verify extraction protocol preserves the protein of interest

• Check if the epitope is masked by protein denaturation conditions

High Background:

• Increase blocking stringency (5% BSA or milk, 0.1% Tween-20)

• Reduce primary antibody concentration

• Increase wash number and duration (5-6 washes, 10 minutes each)

• Filter buffers to remove particulates

• Ensure membranes are fully submerged during all incubations

Multiple Bands:

• Determine if bands represent isoforms, degradation products, or post-translational modifications

• Optimize sample preparation to reduce degradation (add protease inhibitors)

• Compare with positive control samples when available

• Test different lysis buffers for optimal extraction

Document all optimization steps systematically to establish reproducible protocols for future experiments .

How can computational antibody design approaches improve research tools for studying S. pombe proteins like SPAC29A4.23?

Computational tools offer significant advantages for developing optimized antibodies:

RosettaAntibodyDesign (RAbD):

RAbD employs a structural-bioinformatics approach to design antibodies with improved target specificity. The framework:

• Samples diverse sequence and structural space of antibodies

• Models antibody-antigen complexes in customizable protocols

• Grafts structures from canonical clusters of complementarity-determining regions (CDRs)

• Optimizes binding properties through computational prediction of interaction energy

IsAb Protocol:

IsAb provides a computational antibody design pipeline that:

• Predicts 3D antibody structures when experimental structures are unavailable

• Uses RosettaRelax to minimize energy of protein structures

• Performs two-step docking (global and local) to predict binding conformations

• Identifies hotspots through alanine scanning

• Conducts computational affinity maturation to improve antibody properties

Researchers can leverage these tools to design improved antibodies against difficult targets or to enhance the performance of existing antibodies against SPAC29A4.23 .

What considerations are important when using SPAC29A4.23 Antibody to study protein dynamics during cell cycle progression?

Studying protein dynamics throughout the cell cycle requires careful experimental design:

Cell Synchronization:

• Use established methods for S. pombe synchronization (nitrogen starvation, hydroxyurea block, or temperature-sensitive cdc mutants)

• Collect samples at defined intervals (typically 20-30 minute intervals over 4-6 hours)

• Verify synchronization efficiency using DAPI staining and FACS analysis

Protein Analysis:

• Quantify expression by Western blot at each time point

• Normalize to appropriate loading controls (e.g., α-tubulin)

• Track localization changes by immunofluorescence microscopy

• Consider using time-lapse imaging for dynamic studies

Data Integration:

• Correlate protein levels/localization with cell cycle phase markers

• Create quantitative profiles of protein expression throughout the cycle

• Compare behavior under normal and perturbed conditions

How might SPAC29A4.23 Antibody be applied to investigate potential roles in S. pombe cell wall formation?

If SPAC29A4.23 plays a role in cell wall biology, several specialized approaches can be employed:

Localization Studies:

• Immunogold electron microscopy to precisely localize the protein within cell wall layers

• Co-localization with known cell wall synthesis enzymes

• Tracking localization during septum formation and cell division

Functional Analysis:

• Compare expression and localization in wild-type versus cell wall mutants

• Analyze protein behavior under cell wall stress conditions (e.g., calcofluor white, Congo red)

• Investigate protein dynamics during protoplast regeneration

Cell Wall Component Analysis:

• Combine antibody studies with specific staining for cell wall components:

  • Aniline blue for β-1,3-glucan

  • Wheat germ agglutinin for chitin/chitosan

  • Concanavalin A for mannoproteins

• Assess cell wall composition in strains with altered SPAC29A4.23 expression

This multi-faceted approach can reveal connections between SPAC29A4.23 and specific aspects of cell wall architecture or dynamics .

What approaches should researchers use to investigate potential post-translational modifications of SPAC29A4.23?

Identifying and characterizing post-translational modifications requires specialized techniques:

Detection Methods:

• Mobility shift assays on Western blots (modified proteins often migrate differently)

• Modification-specific antibodies (phospho-, glyco-, ubiquitin-specific)

• Stains for glycoproteins (PAS-Silver staining as mentioned in search result 9)

• EndoH treatment to detect N-glycosylation (procedure mentioned in search result 9)

Enrichment Strategies:

• Phosphopeptide enrichment (IMAC, TiO₂ chromatography)

• Glycopeptide enrichment (lectin affinity, hydrazide chemistry)

• Ubiquitinated protein capture (TUBE technology)

Analytical Approaches:

• 2D gel electrophoresis to separate modified isoforms

• Mass spectrometry for precise identification of modifications and their sites

• Targeted analysis based on motif predictions (e.g., phosphorylation motifs)

• Comparison of modification patterns under different cellular conditions

For comprehensive characterization, combining multiple approaches provides the most reliable results .

How can researchers integrate antibody-based detection of SPAC29A4.23 with multi-omics approaches in S. pombe studies?

Modern research benefits from integrating multiple data types:

Integrative Experimental Approaches:

• ChIP-seq: If SPAC29A4.23 interacts with DNA or chromatin, use the antibody for chromatin immunoprecipitation followed by sequencing

• RIP-seq: For RNA-binding proteins, perform RNA immunoprecipitation followed by sequencing

• IP-MS: Combine immunoprecipitation with mass spectrometry to identify interaction partners

Data Integration Strategies:

• Correlate protein expression levels with corresponding gene expression (transcriptomics)

• Map protein interactions to metabolic pathways (metabolomics)

• Relate localization changes to chromosomal organization data (Hi-C)

Analysis Framework:

• Employ computational tools to integrate diverse datasets

• Utilize network analysis to place SPAC29A4.23 in functional contexts

• Develop predictive models based on multi-omics data

This integrative approach provides a systems-level understanding of SPAC29A4.23 function within the broader cellular context, revealing insights not attainable through single-technique approaches .

Resources for S. pombe Research with Antibodies

For researchers working with SPAC29A4.23 Antibody, these resources can provide valuable support: