SPAC26H5.05 Antibody

What is SPAC26H5.05 antibody and what are its primary research applications?

SPAC26H5.05 antibody is a polyclonal antibody raised against Schizosaccharomyces pombe (strain 972 / ATCC 24843, fission yeast) SPAC26H5.05 protein. It is primarily used for:

• ELISA (Enzyme-Linked Immunosorbent Assay): For quantitative detection of the target protein

• Western Blot (WB): For identification of target protein in complex mixtures

• Immunohistochemical analyses: To examine protein expression in tissue samples

The antibody specifically targets Schizosaccharomyces pombe (fission yeast) proteins and is delivered in liquid form with 50% Glycerol in a 0.01M PBS (pH 7.4) buffer with 0.03% Proclin 300 as a preservative .

How does SPAC26H5.05 antibody differ from other yeast-targeted antibodies?

SPAC26H5.05 antibody specifically targets a yeast protein in Schizosaccharomyces pombe, whereas other yeast antibodies may target different proteins or different yeast species. Key differentiating factors include:

The specificity of the antibody is critical for research applications focusing on S. pombe models, which are widely used for studying cell cycle, DNA damage repair, and other conserved cellular processes .

What are the optimal protocols for using SPAC26H5.05 antibody in Western blot applications?

For optimal Western blot results with SPAC26H5.05 antibody, follow this validated protocol:

• Sample preparation:

  • Lyse S. pombe cells in an appropriate buffer (e.g., RIPA buffer with protease inhibitors)

  • Determine protein concentration using Bradford or BCA assay

  • Prepare 20-50 μg of total protein per lane

• Gel electrophoresis and transfer:

  • Separate proteins using 10-12% SDS-PAGE

  • Transfer to PVDF or nitrocellulose membrane (0.45 μm pore size)

• Blocking and antibody incubation:

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

  • Dilute SPAC26H5.05 antibody at 1:500-1:2000 in blocking buffer

  • Incubate overnight at 4°C with gentle rocking

• Secondary antibody incubation:

  • Wash membrane 3× with TBST, 5 minutes each

  • Incubate with goat anti-rabbit IgG-HRP (1:5000) for 1 hour at room temperature

  • Wash 3× with TBST, 5 minutes each

• Detection:

  • Apply ECL substrate and detect signal using film or digital imaging system

For optimal signal-to-noise ratio, titration of both primary and secondary antibodies is recommended .

What are the recommended conditions for ELISA using SPAC26H5.05 antibody?

For ELISA applications with SPAC26H5.05 antibody, follow this optimized protocol:

• Plate coating:

  • Coat plates with target antigen (1-10 μg/ml) in carbonate buffer (pH 9.6)

  • Incubate overnight at 4°C

• Blocking:

  • Block with 1-3% BSA or 5% non-fat milk in PBS for 1-2 hours at room temperature

• Primary antibody:

  • Dilute SPAC26H5.05 antibody at 1:500-1:5000 in blocking buffer

  • Incubate for 1-2 hours at room temperature

• Secondary antibody:

  • Wash 3-5× with PBST

  • Apply HRP-conjugated goat anti-rabbit IgG at 1:5000-1:10000 dilution

  • Incubate for 1 hour at room temperature

• Detection:

  • Wash 3-5× with PBST

  • Add TMB substrate and incubate for 5-30 minutes at room temperature

  • Stop reaction with 2N H₂SO₄ and read at 450 nm

For accurate quantification, include a standard curve using purified recombinant protein .

How can SPAC26H5.05 antibody be utilized in high-throughput screening of yeast mutant libraries?

SPAC26H5.05 antibody can be effectively integrated into high-throughput screening protocols for yeast mutant libraries:

• Array-based screening platform setup:

  • Grow yeast colonies in 96 or 384-well format

  • Transfer to nitrocellulose membranes using robotic spotters

• Antibody screening process:

  • Lyse cells directly on membranes using alkaline lysis

  • Block with 5% BSA in TBST

  • Incubate with SPAC26H5.05 antibody (1:1000)

  • Detect with secondary antibody and chemiluminescent substrate

• Data analysis workflow:

  • Image using high-resolution scanner

  • Quantify signal intensity using image analysis software

  • Normalize against control spots

  • Apply statistical threshold for hit identification

• Validation strategy:

  • Confirm hits with individual Western blots

  • Perform secondary assays to validate functional relevance

This approach allows screening of thousands of mutants simultaneously, with a recent study identifying 676 potential interacting clonotypes using a similar high-throughput antibody screening approach .

What are the considerations for using SPAC26H5.05 antibody in immunoprecipitation experiments with fission yeast extracts?

When using SPAC26H5.05 antibody for immunoprecipitation (IP) of proteins from fission yeast, consider these critical factors:

• Lysis buffer optimization:

  • Use gentle lysis buffer for membrane proteins (50 mM HEPES pH 7.5, 150 mM NaCl, 1% NP-40, 0.1% SDS, protease inhibitors)

  • For nuclear proteins, include DNase treatment to reduce viscosity

• Pre-clearing strategy:

  • Pre-clear lysate with Protein A beads to reduce nonspecific binding

  • Incubate lysate with 1-5 μg SPAC26H5.05 antibody per 500 μg total protein

  • Add Protein A beads and incubate 2-4 hours at 4°C with rotation

• Washing conditions:

  • Perform 4-5 stringent washes with lysis buffer

  • Consider adding increasing salt concentrations (150-500 mM NaCl) for final washes to reduce nonspecific binding

• Controls to include:

  • IgG control from the same species (rabbit)

  • Input sample (5-10% of starting material)

  • Unbound fraction sample

• Elution methods:

  • Gentle elution with low pH glycine buffer (100 mM, pH 2.5)

  • Alternative: SDS sample buffer at 95°C for 5 minutes

Success in IP experiments often requires extensive optimization, with affinity-purification coupled to mass spectrometry providing a powerful approach for identifying protein interactions .

What are common issues with non-specific binding of SPAC26H5.05 antibody and how can they be addressed?

Non-specific binding is a common challenge when working with polyclonal antibodies like SPAC26H5.05. Here are methodological approaches to address this issue:

• Enhanced blocking protocols:

  • Extend blocking time to 2-3 hours at room temperature

  • Test alternative blocking agents (5% BSA, 5% casein, commercial blocking buffers)

  • Add 0.1-0.5% Tween-20 to blocking buffer to reduce hydrophobic interactions

• Antibody dilution optimization:

  • Test serial dilutions (1:500, 1:1000, 1:2000, 1:5000)

  • Prepare antibody in fresh blocking buffer

  • Consider overnight incubation at 4°C to improve specificity

• Washing optimization:

  • Increase washing stringency (add up to 0.1% SDS to wash buffer)

  • Extend washing times (5 washes, 10 minutes each)

  • Use higher salt concentration (up to 500 mM NaCl) in wash buffer

• Pre-adsorption technique:

  • Incubate antibody with non-target protein extract (e.g., from deletion strains)

  • Dilute the pre-adsorbed antibody to working concentration

  • Apply to experimental samples

• Affinity purification of antibody:

  • Immobilize target antigen on column

  • Pass antibody solution through column

  • Elute specifically bound antibodies

These approaches have significantly reduced non-specific binding in controlled experiments, with up to 85% reduction in background signal demonstrated in similar polyclonal antibody applications .

How can epitope mapping be performed to validate SPAC26H5.05 antibody specificity?

Epitope mapping is crucial for validating antibody specificity. For SPAC26H5.05 antibody, consider these methodological approaches:

• Peptide array screening:

  • Synthesize overlapping peptides (15-20 amino acids) spanning the SPAC26H5.05 protein

  • Spot peptides on membrane or glass slide

  • Probe with SPAC26H5.05 antibody followed by HRP-conjugated secondary antibody

  • Identify reactive peptides indicating epitope regions

• Alanine scanning mutagenesis:

  • Generate mutants where each amino acid is systematically replaced with alanine

  • Express and purify mutant proteins

  • Test antibody binding by ELISA or Western blot

  • Identify residues critical for antibody recognition

• Molecular docking and computational prediction:

  • Generate 3D structural models using AlphaFold2

  • Perform molecular docking to predict antibody-antigen binding sites

  • Validate predictions experimentally

• Mass spectrometry approaches:

  • Perform hydrogen-deuterium exchange mass spectrometry

  • Compare deuterium uptake in free protein versus antibody-bound protein

  • Identify protected regions indicating epitope location

Research has shown that combining computational prediction with experimental validation provides the most accurate epitope mapping results, with approaches similar to those used for SpA5 epitope identification in recent studies .

How does the specificity and sensitivity of SPAC26H5.05 antibody compare to monoclonal antibodies targeting similar epitopes?

SPAC26H5.05 antibody (polyclonal) versus monoclonal antibodies targeting similar epitopes:

What are the advantages and limitations of using SPAC26H5.05 antibody compared to genetic tagging approaches for protein detection?

Comparison between SPAC26H5.05 antibody-based detection and genetic tagging:

How can SPAC26H5.05 antibody be adapted for single-cell analysis techniques in yeast research?

Adapting SPAC26H5.05 antibody for single-cell analysis requires specialized methodologies:

• Flow cytometry optimization:

  • Fix yeast cells with 3.7% formaldehyde for 30 minutes

  • Digest cell wall with zymolyase or lyticase

  • Permeabilize with 0.1% Triton X-100

  • Block with 3% BSA in PBS

  • Incubate with SPAC26H5.05 antibody (1:100-1:500)

  • Apply fluorophore-conjugated secondary antibody (e.g., goat anti-rabbit IgG)

  • Include controls: unstained cells, secondary-only, isotype control

• Single-cell immunofluorescence microscopy:

  • Grow cells on concanavalin A-coated slides

  • Fix with 4% paraformaldehyde

  • Permeabilize cell wall with 1.2 M sorbitol + zymolyase

  • Block with 3% BSA in PBS for 1 hour

  • Incubate with SPAC26H5.05 antibody overnight at 4°C

  • Apply fluorescent secondary antibody for 1 hour at room temperature

  • Counterstain nuclei with DAPI

• Mass cytometry (CyTOF) adaptation:

  • Conjugate SPAC26H5.05 antibody with metal isotopes

  • Perform barcoding for multiplexed analysis

  • Analyze using standard CyTOF protocols

• Microfluidic-based single-cell Western blot:

  • Capture single cells in microfluidic chambers

  • Perform in situ lysis, protein separation, and antibody probing

  • Analyze protein expression at single-cell level

Single-cell approaches provide insights into cell-to-cell variability and have been successfully applied in recent antibody repertoire analysis studies using microfluidic platforms .

What considerations should be made when using SPAC26H5.05 antibody for chromatin immunoprecipitation experiments in fission yeast?

For chromatin immunoprecipitation (ChIP) applications with SPAC26H5.05 antibody, consider these methodological requirements:

• Crosslinking optimization:

  • Test different formaldehyde concentrations (1-3%)

  • Optimize crosslinking time (5-20 minutes)

  • Quench with glycine (125 mM final concentration)

• Chromatin preparation:

  • Lyse cells using glass beads or enzymatic methods

  • Sonicate to fragment chromatin (200-500 bp fragments)

  • Verify fragment size by agarose gel electrophoresis

• Immunoprecipitation conditions:

  • Pre-clear chromatin with Protein A beads

  • Use 3-5 μg SPAC26H5.05 antibody per 25-100 μg chromatin

  • Include appropriate controls:

    • Input chromatin (10%)

    • Non-specific IgG control

    • Positive control (known target)

• Washing and elution:

  • Perform sequential washes with increasing stringency

  • Elute protein-DNA complexes with elution buffer

  • Reverse crosslinks (65°C overnight)

  • Purify DNA using column-based methods

• Analysis methods:

  • qPCR for targeted analysis

  • Next-generation sequencing for genome-wide profiling

The success of ChIP experiments heavily depends on whether the target protein is directly or indirectly associated with DNA and whether the antibody can access the epitope in a crosslinked chromatin environment .

How might computational antibody design techniques be applied to improve SPAC26H5.05 antibody specificity or affinity?

Computational approaches offer promising avenues for enhancing SPAC26H5.05 antibody:

• Structure-based antibody engineering:

  • Generate 3D models of the antibody using AlphaFold2

  • Perform molecular docking with the target antigen

  • Identify key binding residues through computational alanine scanning

  • Design targeted mutations to improve binding affinity

• In silico affinity maturation:

  • Implement Rosetta-based antibody design protocols

  • Simulate multiple rounds of somatic hypermutation

  • Score and rank mutant candidates

  • Validate top candidates experimentally

• Epitope-focused antibody design:

  • Identify conserved epitopes across related proteins

  • Design antibodies specifically targeting unique epitopes

  • Reduce cross-reactivity through negative design principles

• Machine learning approaches:

  • Train models on antibody-antigen binding data

  • Predict binding affinity and specificity for novel designs

  • Implement deep learning for sequence-based optimization

The IsAb computational protocol has demonstrated success in antibody design, with key steps including antigen modeling, epitope prediction, antibody modeling, docking, refinement, and affinity maturation .

What potential exists for using SPAC26H5.05 antibody in multiplexed detection systems for studying protein-protein interactions in fission yeast?

SPAC26H5.05 antibody can be incorporated into advanced multiplexed detection systems:

• Multiplex immunofluorescence approaches:

  • Sequential staining with multiple antibodies

  • Use spectrally distinct fluorophores for each antibody

  • Apply tyramide signal amplification for enhanced sensitivity

  • Employ multispectral imaging for signal separation

• Proximity ligation assay (PLA) applications:

  • Combine SPAC26H5.05 with antibodies against potential interaction partners

  • Use species-specific PLA probes with attached oligonucleotides

  • Amplify signal only when proteins are in close proximity (<40 nm)

  • Visualize interactions as distinct puncta

• Mass cytometry adaptations:

  • Label multiple antibodies with distinct metal isotopes

  • Simultaneously detect >40 proteins in single cells

  • Apply dimensional reduction algorithms for data analysis

• Spatial transcriptomics integration:

  • Combine protein detection with RNA localization

  • Correlate protein expression with transcriptional state

  • Create spatial maps of protein-RNA relationships

Recent developments in single-cell protein analysis have enabled the construction of protein interaction networks with unprecedented resolution, with one study identifying 10 pairs of highly expressed clonal immunoglobulin genes using next-generation sequencing approaches coupled with antibody profiling .

What are the optimal storage conditions for maintaining SPAC26H5.05 antibody activity over extended periods?

For maximum stability and activity retention of SPAC26H5.05 antibody:

• Short-term storage (up to 1 month):

  • Store at 2-8°C in the original container

  • Avoid repeated freeze-thaw cycles

  • Keep away from direct light

• Long-term storage (>1 month):

  • Store at -20°C to -80°C in small working aliquots

  • Add stabilizing proteins if needed (e.g., 0.1% BSA)

  • Use glycerol (50% final concentration) to prevent freezing damage

• Handling recommendations:

  • Allow antibody to equilibrate to room temperature before opening

  • Centrifuge briefly before opening to collect all liquid

  • Return to storage immediately after use

  • Avoid contamination by using clean pipette tips

• Monitoring stability:

  • Record date of first use and thawing events

  • Periodically test reactivity against positive controls

  • Compare signal intensity over time

  • Consider preparing a standard curve for quantitative applications

Under optimal storage conditions, SPAC26H5.05 antibody typically maintains activity for at least 12 months, though performance should be validated before critical experiments .

What quality control measures should be implemented when working with different batches of SPAC26H5.05 antibody?

To ensure experimental consistency across different antibody batches:

• Incoming batch validation:

  • Perform side-by-side Western blot comparison with previous batch

  • Establish minimum acceptable signal-to-noise ratio

  • Verify specific band detection at expected molecular weight

  • Document batch-specific optimal dilutions

• Reference standard preparation:

  • Create a large batch of positive control (target protein extract)

  • Aliquot and store at -80°C

  • Use as reference for all new antibody batches

• Quantitative performance metrics:

  • Generate standard curves for each batch

  • Determine limit of detection and linear range

  • Calculate coefficient of variation between technical replicates

  • Compare EC50 values across batches

• Documentation and record-keeping:

  • Maintain detailed batch records including:

    • Manufacturer lot number

    • Date received and opened

    • Initial validation results

    • Optimization parameters for specific applications

    • Any observed anomalies

• Cross-referencing with orthogonal methods:

  • Validate critical findings with alternative detection methods

  • Consider genetic approaches (e.g., tagging) for key experiments

Implementing these measures can significantly reduce experimental variability, with studies showing that proper validation can reduce inter-batch coefficient of variation from >30% to <10% .