SPAC23H3.11c Antibody

What is SPAC23H3.11c and what is its function in Schizosaccharomyces pombe?

SPAC23H3.11c is a protein expressed in Schizosaccharomyces pombe (fission yeast). Based on current research, this protein is required for the synthesis of major beta-glucans in the yeast cell wall . Understanding this protein's function has implications for fundamental research in cell wall biosynthesis and maintenance, which is critical for cellular integrity in fungi.

The protein belongs to a group of gene products that may be involved in heterochromatin regulation networks, as other proteins in similar regions (such as those mentioned in search result ) interact with chromatin modifying complexes. For example, research has shown that proteins in similar chromosomal regions are upregulated during specific conditions such as meiosis .

What types of SPAC23H3.11c antibodies are currently available for research?

Currently, polyclonal antibodies against SPAC23H3.11c are available for research purposes. These are primarily:

• Rabbit anti-Schizosaccharomyces pombe SPAC23H3.11c Polyclonal Antibodies

• The antibodies are typically raised against recombinant Schizosaccharomyces pombe (strain 972/ATCC 24843) SPAC23H3.11c protein

These antibodies are supplied in liquid form with specific storage buffer compositions:

• Preservative: 0.03% Proclin 300

• Constituents: 50% Glycerol, 0.01M PBS, pH 7.4

They are designed for research applications including ELISA and Western Blot analyses, specifically focusing on the identification of the target antigen in experimental systems.

How should researchers properly store and handle SPAC23H3.11c antibodies?

Proper storage and handling of SPAC23H3.11c antibodies is crucial for maintaining their efficacy:

Storage Recommendations:

• Upon receipt, store at -20°C or -80°C

• Avoid repeated freeze-thaw cycles as these can damage antibody structure and function

• Small volumes of antibody may occasionally become entrapped in the seal of the product vial during shipment and storage

• If necessary, briefly centrifuge the vial on a tabletop centrifuge to dislodge any liquid in the container's cap

Handling Guidelines:

• Always wear appropriate personal protective equipment

• Allow antibodies to equilibrate to room temperature before opening

• When diluting, use appropriate buffers as recommended in the product documentation

• Prepare working aliquots to minimize freeze-thaw cycles

• Document lot numbers, receipt dates, and usage to track antibody performance

What are the validated applications for SPAC23H3.11c antibodies?

Based on available data, SPAC23H3.11c antibodies have been validated for the following applications:

Researchers should note that these applications have been specifically tested to ensure identification of the target antigen . Additional applications may require further validation by the end-user.

How can researchers validate the specificity of SPAC23H3.11c antibodies in their experimental systems?

Comprehensive Validation Protocol:

• Positive and Negative Controls:

  • Use wild-type S. pombe strains (strain 972/ATCC 24843) as positive controls

  • Use SPAC23H3.11c deletion mutants or knockdown strains as negative controls

  • Include closely related species as specificity controls

• Western Blot Validation:

  • Confirm single band of expected molecular weight (check predicted MW of SPAC23H3.11c)

  • Run parallel blots with pre-immune serum or isotype controls

  • Perform peptide competition assays by pre-incubating antibody with excess recombinant SPAC23H3.11c protein

• Immunoprecipitation Followed by Mass Spectrometry:

  • Perform IP using the antibody

  • Analyze precipitated proteins by mass spectrometry

  • Confirm presence of SPAC23H3.11c and assess off-target binding

• Cross-validation with Tagged Protein:

  • Generate S. pombe strains expressing tagged SPAC23H3.11c (GFP, YFP, or epitope tags)

  • Compare localization/detection patterns between antibody and tag detection

  • Similar to approaches used for other S. pombe proteins such as Epe1-YFP validation

• Genetic Approaches:

  • Test antibody reactivity in strains with varying expression levels of SPAC23H3.11c

  • Use inducible systems to demonstrate correlation between expression and signal intensity

What are the optimal protocols for SPAC23H3.11c antibody use in chromatin immunoprecipitation (ChIP) assays?

While specific ChIP protocols for SPAC23H3.11c are not directly described in the provided search results, researchers can adapt protocols used for similar S. pombe proteins such as Epe1:

Optimized ChIP Protocol for S. pombe Proteins:

• Cell Culture and Crosslinking:

  • Grow S. pombe cultures in appropriate media (e.g., YES media) to logarithmic phase

  • Harvest cells by centrifugation at 4°C

  • Crosslink protein-DNA interactions with 1% formaldehyde for 10 minutes at room temperature

  • Quench with 125 mM glycine

• Cell Lysis and Chromatin Preparation:

  • Disrupt cells using a bead beater (e.g., Fastprep FP120) at speed 6.5, five times for 20 seconds

  • Extract chromatin following established S. pombe protocols

  • Sonicate to generate DNA fragments of 200-500 bp

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads

  • Incubate cleared chromatin with SPAC23H3.11c antibody (2-5 μg) overnight at 4°C

  • Include appropriate controls (IgG, no antibody)

  • Capture antibody-chromatin complexes with protein A/G beads

  • Wash extensively with increasingly stringent buffers

• DNA Recovery and Analysis:

  • Reverse crosslinks (65°C overnight)

  • Treat with RNase A and Proteinase K

  • Purify DNA using commercial kits

  • Analyze by qPCR, sequencing, or other appropriate methods

• Data Analysis:

  • Calculate enrichment relative to input and control antibody

  • Normalize using appropriate reference genes

  • Correct for the ratios in whole-cell extracts (WCE) as done in similar studies

How can researchers optimize Western blot protocols for detection of SPAC23H3.11c in S. pombe samples?

Optimized Western Blot Protocol:

• Sample Preparation:

  • Harvest S. pombe cells during logarithmic growth phase

  • Prepare whole cell extracts using mechanical disruption (glass beads or bead beater)

  • Extract proteins in appropriate lysis buffer containing protease inhibitors

  • Quantify protein concentration using Bradford or BCA assay

• Gel Electrophoresis:

  • Use 10-12% SDS-PAGE gels (adjust based on SPAC23H3.11c molecular weight)

  • Load 20-50 μg of total protein per lane

  • Include molecular weight markers

  • Run at constant voltage (100-120V) until adequate separation

• Transfer Optimization:

  • Use PVDF membrane (0.45 μm) for standard applications

  • Transfer at constant current (250-300 mA) for 60-90 minutes

  • Verify transfer efficiency with reversible staining (Ponceau S)

• Blocking and Antibody Incubation:

  • Block with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

  • Incubate with SPAC23H3.11c antibody (typical starting dilution 1:1000) overnight at 4°C

  • Wash 3x with TBST, 10 minutes each

  • Incubate with secondary antibody (anti-rabbit HRP, 1:5000) for 1 hour at room temperature

  • Wash 4x with TBST, 10 minutes each

• Detection and Analysis:

  • Develop using ECL substrate compatible with your imaging system

  • Expose to film or capture using digital imaging system

  • Quantify band intensity using appropriate software (e.g., Fujifilm Image Gauge software)

  • Normalize to appropriate loading control (e.g., SPAPB1A10.11c)

• Troubleshooting Tips:

  • If high background occurs, increase blocking time or washing stringency

  • If weak signal occurs, increase antibody concentration or incubation time

  • For non-specific bands, optimize antibody dilution and consider pre-absorbing with non-specific proteins

What are the methodological considerations for using SPAC23H3.11c antibodies in immunofluorescence microscopy studies?

Optimized Immunofluorescence Protocol for S. pombe:

• Cell Preparation:

  • Grow S. pombe cells to mid-log phase

  • Fix with 3.7% formaldehyde for 30 minutes at room temperature

  • Wash cells in PEM buffer (100 mM PIPES, 1 mM EGTA, 1 mM MgSO₄, pH 6.9)

  • Digest cell wall with zymolyase (1 mg/ml) in PEMS (PEM + 1.2 M sorbitol) for 30-60 minutes

  • Permeabilize with 1% Triton X-100 in PEM for 1 minute

• Antibody Staining:

  • Block with PEMBAL (PEM + 1% BSA, 0.1% sodium azide, 100 mM lysine hydrochloride) for 30 minutes

  • Incubate with primary SPAC23H3.11c antibody (1:100-1:500 dilution in PEMBAL) overnight at 4°C

  • Wash 3x with PEMBAL

  • Incubate with fluorophore-conjugated secondary antibody (1:500 in PEMBAL) for 2 hours at room temperature

  • Wash 3x with PEMBAL

  • Mount with anti-fade mounting medium containing DAPI

• Controls and Validation:

  • Include negative controls (no primary antibody, pre-immune serum)

  • Use strains expressing fluorescently tagged SPAC23H3.11c for co-localization studies

  • Consider similar approaches used for other S. pombe proteins such as Epe1-YFP

• Imaging and Analysis:

  • Capture images using confocal or widefield fluorescence microscopy

  • Analyze protein localization patterns (e.g., nuclear, cytoplasmic, or specific foci)

  • Quantify signal intensity and distribution using appropriate image analysis software

• Expected Results:

  • Based on similar proteins, researchers might look for specific nuclear foci or diffuse patterns

  • Validate localization patterns in response to different growth conditions

  • Consider examining localization dependencies on other proteins (similar to how Epe1 localization depends on Swi6)

How can researchers design experiments to investigate potential interactions between SPAC23H3.11c and other S. pombe proteins?

Experimental Design Framework:

• Co-Immunoprecipitation (Co-IP):

  • Generate S. pombe lysates under non-denaturing conditions

  • Perform IP with SPAC23H3.11c antibody

  • Analyze co-precipitated proteins by Western blot or mass spectrometry

  • Include appropriate controls (IgG, lysate from deletion strains)

  • Validate interactions with reciprocal Co-IP experiments

• Two-Hybrid Assays:

  • Similar to approaches used for other S. pombe proteins (e.g., Swi6 and Epe1 interactions)

  • Clone SPAC23H3.11c into yeast two-hybrid bait vectors

  • Screen against S. pombe cDNA library or candidate interactors

  • Validate positive interactions with targeted assays

• Proximity Labeling:

  • Generate strains expressing SPAC23H3.11c fused to BioID or APEX2

  • Induce proximity labeling in living cells

  • Purify biotinylated proteins and identify by mass spectrometry

  • Validate interactions using orthogonal methods

• Genetic Interaction Mapping:

  • Create double mutants combining SPAC23H3.11c deletion with other gene deletions

  • Analyze genetic interactions using epistasis analysis

  • Look for synthetic phenotypes that suggest functional relationships

  • Consider global approaches like synthetic genetic array (SGA) analysis

• Fluorescence Microscopy Approaches:

  • Create strains expressing fluorescently tagged SPAC23H3.11c and candidate interactors

  • Analyze co-localization patterns under different conditions

  • Consider advanced techniques like FRET or FCCS to detect direct interactions

  • Similar to fluorescence microscopy approaches used for Epe1-YFP and Swi6

What approaches can researchers use to investigate post-translational modifications of SPAC23H3.11c?

Comprehensive PTM Investigation Strategy:

• Mass Spectrometry Analysis:

  • Immunoprecipitate SPAC23H3.11c from S. pombe lysates

  • Perform tryptic digestion and LC-MS/MS analysis

  • Search for common PTMs (phosphorylation, acetylation, methylation, ubiquitination)

  • Use enrichment methods specific for certain PTMs (e.g., TiO₂ for phosphopeptides)

• Western Blot Analysis:

  • Use antibodies specific for common PTMs

  • Perform IP with SPAC23H3.11c antibody, then probe with PTM-specific antibodies

  • Compare PTM patterns under different growth conditions or genetic backgrounds

  • Use appropriate controls (phosphatase treatment, deacetylase treatment)

• Genetic Approaches:

  • Generate mutants with predicted PTM sites altered (e.g., S→A for phosphorylation sites)

  • Assess functional consequences of mutations

  • Create deletion/knockout strains of enzymes predicted to modify SPAC23H3.11c

  • Assess changes in SPAC23H3.11c function or localization

• Inhibitor Studies:

  • Treat cells with inhibitors of specific PTM enzymes

  • Assess changes in SPAC23H3.11c function, localization, or interaction network

  • Consider both general inhibitors and S. pombe-specific approaches

• Functional Validation:

  • Express modified forms of SPAC23H3.11c in deletion backgrounds

  • Assess complementation of phenotypes

  • Compare cellular responses under different stress conditions

  • Analyze changes in protein-protein interactions

How can researchers troubleshoot non-specific binding when using SPAC23H3.11c antibodies?

Systematic Troubleshooting Strategy:

• Identify the Problem:

  • Multiple bands on Western blot

  • Unexpected localization patterns in IF

  • High background in all applications

  • Positive signal in negative controls

• Optimize Blocking Conditions:

  • Test different blocking agents (BSA, milk, commercial blockers)

  • Increase blocking time or concentration

  • Add non-ionic detergents (Tween-20, Triton X-100) to reduce hydrophobic interactions

  • Consider specialized blocking agents for problematic samples

• Antibody Dilution Optimization:

  • Perform titration experiments to determine optimal concentration

  • Typically start with manufacturer's recommendation and adjust as needed

  • For Western blots, consider dilutions from 1:500 to 1:5000

  • For IF, consider dilutions from 1:50 to 1:500

• Pre-absorption Strategies:

  • Incubate antibody with lysates from deletion strains or other species

  • Use recombinant proteins or peptides not containing the epitope

  • Consider commercial pre-absorption kits

• Alternative Detection Methods:

  • Try different secondary antibodies or detection systems

  • Consider more specific detection methods (monoclonal vs. polyclonal)

  • Use protein/epitope tags as alternative approaches

  • Apply proximity ligation assay (PLA) for greater specificity

What techniques can researchers use to enhance signal detection when working with low-abundance SPAC23H3.11c protein?

Signal Enhancement Strategies:

• Sample Preparation Optimization:

  • Enrich for subcellular fractions where SPAC23H3.11c is expected

  • Use gentle lysis methods to preserve protein integrity

  • Add protease and phosphatase inhibitors to prevent degradation

  • Concentrate samples using TCA precipitation or similar methods

• Immunoprecipitation Before Analysis:

  • Perform IP to concentrate SPAC23H3.11c before Western blot

  • Use high-affinity capture methods (e.g., protein A/G magnetic beads)

  • Optimize elution conditions to maximize recovery

• Signal Amplification Methods:

  • Use tyramide signal amplification (TSA) for immunofluorescence

  • Apply polymer-based detection systems for immunohistochemistry

  • Consider biotin-streptavidin amplification systems

  • Use high-sensitivity ECL substrates for Western blot

• Instrument Settings Optimization:

  • Increase exposure time for Western blots

  • Optimize PMT settings for confocal microscopy

  • Use sensitive cameras and longer exposure times for fluorescence imaging

  • Apply deconvolution algorithms to improve signal-to-noise ratio

• Consider Expression Induction:

  • If possible, induce expression of SPAC23H3.11c using appropriate conditions

  • Research conditions that naturally upregulate the protein

  • Similar to approaches used for other S. pombe proteins whose expression is condition-dependent

How can researchers address batch-to-batch variability when using SPAC23H3.11c antibodies?

Managing Antibody Variability:

• Establish Validation Protocols:

  • Develop standardized validation tests for each new antibody batch

  • Create a reference sample set for quality control testing

  • Document performance metrics for each batch (sensitivity, specificity, background)

  • Maintain detailed records of validation results

• Batch Reservation Strategy:

  • Purchase larger quantities of a validated batch for long-term studies

  • Aliquot and store properly to maintain stability

  • Consider negotiating with manufacturers for batch reservation

• Internal Reference Standards:

  • Create standardized positive control samples

  • Use consistent loading controls for Western blots

  • Develop calibration curves for quantitative applications

  • Normalize results to account for batch differences

• Parallel Testing:

  • Test new batches alongside previously validated batches

  • Document differences in performance characteristics

  • Establish correction factors if necessary

  • Consider using multiple antibodies targeting different epitopes

• Long-term Solutions:

  • Consider developing recombinant antibodies for consistent performance

  • Investigate alternatives like nanobodies or aptamers

  • Develop tagged constructs that can be detected with highly standardized antibodies

  • Share validation data with manufacturers to improve product consistency

How can SPAC23H3.11c antibodies be integrated into large-scale proteomic studies of S. pombe?

Integration Strategies for Proteomics:

• Antibody-Based Enrichment Prior to Mass Spectrometry:

  • Use SPAC23H3.11c antibodies for immunoprecipitation

  • Enrich for protein complexes containing SPAC23H3.11c

  • Apply to samples from different growth conditions or genetic backgrounds

  • Analyze by LC-MS/MS to identify interaction partners and modifications

• Reverse Phase Protein Arrays (RPPA):

  • Spot samples from multiple conditions or strains on arrays

  • Probe with SPAC23H3.11c antibody

  • Quantify expression changes across conditions

  • Integrate with other proteomic datasets

• Proximity-Dependent Labeling:

  • Create SPAC23H3.11c fusion with BioID, APEX2, or TurboID

  • Identify proximal proteins through biotinylation

  • Map protein interaction networks under different conditions

  • Validate interactions using SPAC23H3.11c antibodies

• Chromatin Proteomics:

  • If SPAC23H3.11c associates with chromatin, use for ChIP-MS studies

  • Identify co-factors at specific genomic loci

  • Integrate with ChIP-seq data for functional correlation

  • Similar to approaches used for chromatin-associated proteins like Epe1

• Data Integration:

  • Combine antibody-based data with global proteomics datasets

  • Correlate with transcriptomics data to identify regulatory relationships

  • Map onto known protein interaction networks

  • Use bioinformatics to predict functional relationships

What considerations should be taken when developing custom modifications to SPAC23H3.11c antibodies for specialized applications?

Customization Considerations:

• Antibody Fragmentation:

  • Generate Fab or F(ab')₂ fragments for reduced background in certain applications

  • Consider pepsin or papain digestion followed by purification

  • Test fragment functionality in your specific application

  • Optimize concentration due to potential changes in binding characteristics

• Conjugation Strategies:

  • Direct conjugation to fluorophores for single-step detection

  • Biotin conjugation for streptavidin-based amplification

  • Enzyme conjugation (HRP, AP) for direct detection

  • Consider site-specific conjugation methods to preserve binding sites

• Immobilization for Research Tools:

  • Couple to affinity resins for immunoprecipitation or protein purification

  • Attach to sensor surfaces for SPR or BLI studies

  • Immobilize on beads for pull-down assays

  • Optimize orientation to maintain binding capacity

• Recombinant Modifications:

  • Consider developing recombinant versions with standardized formats

  • Explore single-chain variable fragments (scFvs) for smaller size

  • Investigate nanobody or VHH formats for specialized applications

  • Design fusion proteins with functional domains for specific purposes

• Quality Control Considerations:

  • Validate activity after each modification step

  • Determine optimal storage conditions for modified antibodies

  • Assess shelf-life of modified preparations

  • Document batch-to-batch consistency of modification process

How can researchers develop CRISPR-based approaches to complement antibody studies of SPAC23H3.11c?

CRISPR-Based Complementary Strategies:

• Endogenous Tagging:

  • Design CRISPR-Cas9 strategy to insert fluorescent or epitope tags at the SPAC23H3.11c locus

  • Use established S. pombe CRISPR protocols with appropriate modifications

  • Verify tag integration by PCR and sequencing

  • Validate protein expression and function after tagging

• Functional Genomics:

  • Create CRISPR knockout or knockdown of SPAC23H3.11c

  • Apply CRISPR interference (CRISPRi) for tunable repression

  • Develop CRISPR activation (CRISPRa) for overexpression studies

  • Compare phenotypes with antibody-based functional inhibition

• Domain Analysis:

  • Use CRISPR to create specific domain deletions or mutations

  • Target conserved regions identified through bioinformatics

  • Analyze effects on protein function, localization, and interactions

  • Validate with antibody-based detection of modified proteins

• Regulome Analysis:

  • Apply CRISPR screens to identify regulators of SPAC23H3.11c

  • Use CRISPRi library to identify factors affecting SPAC23H3.11c expression

  • Combine with antibody-based quantification of SPAC23H3.11c levels

  • Validate hits with targeted experiments

• Live Cell Studies:

  • Create CRISPR knock-in of split fluorescent proteins

  • Develop CRISPR-based optogenetic controllers of SPAC23H3.11c

  • Apply CRISPR-based proximity labeling systems

  • Complement with fixed-cell antibody studies for validation