SPAPB24D3.06c Antibody

What is SPAPB24D3.06c and what is its significance in S. pombe research?

SPAPB24D3.06c is a protein found in Schizosaccharomyces pombe (fission yeast), identified with UniProt number Q9C0Y8 . It appears to be classified as a sequence orphan in the S. pombe genome, meaning its function is not clearly characterized through sequence homology. The protein's significance lies in its potential involvement in chromatin-associated processes, as it has been identified in proteomic analyses of chromatin-bound proteins .

Current research suggests potential roles in:

• DNA repair pathways (possible relation to other SPAPB locus proteins)

• Gene expression regulation, particularly in stress responses

• Potential involvement in antisense lncRNA-mediated regulation mechanisms

What are the key specifications of commercially available SPAPB24D3.06c antibodies?

The primary commercially available antibody is a rabbit polyclonal antibody with the following specifications:

Standard components typically include :

• 200μg antigens (positive control)

• 1ml pre-immune serum (negative control)

• Purified rabbit polyclonal antibodies

How should SPAPB24D3.06c antibody be used in Western blot analyses?

For optimal Western blot results with SPAPB24D3.06c antibody, follow this methodological approach:

Sample Preparation:

• Grow S. pombe cells to mid-log phase (OD595 0.5)

• Harvest cells and lyse using glass beads with 3 × 30-second pulses in a bead beater at 4°C

• Prepare lysate in appropriate buffer (e.g., 50 mM HEPES pH 7.5, 120 mM KCl, 5 mM EDTA, 0.1% NP-40, 10% glycerol with protease inhibitors)

• Determine protein concentration using Bradford assay

SDS-PAGE and Transfer:

• Load 30μg protein per lane on 12% SDS-PAGE gel

• Transfer to nitrocellulose or PVDF membrane

• Verify transfer with Ponceau S staining

Immunodetection:

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

• Incubate with SPAPB24D3.06c antibody at optimized dilution (typically 1:1000 to 1:5000) overnight at 4°C

• Wash membrane 3× with TBST

• Incubate with HRP-conjugated anti-rabbit secondary antibody (1:5000) for 1 hour

• Wash 3× with TBST

• Develop using ECL substrate and detect using appropriate imaging system

Critical Controls:

• Positive control: S. pombe strain expressing tagged SPAPB24D3.06c (if available)

• Negative control: Untagged wild-type strain to assess non-specific binding

• Loading control: Anti-α-tubulin antibody for normalization

How can SPAPB24D3.06c antibody be optimized for Chromatin Immunoprecipitation (ChIP) experiments?

While not explicitly validated for ChIP in product specifications, researchers can optimize SPAPB24D3.06c antibody for ChIP experiments based on established protocols from related antibodies in S. pombe:

Experimental Protocol:

• Crosslinking: Treat S. pombe cells with 1% formaldehyde for 10 minutes at room temperature

• Quenching: Add glycine (0.4M final) for 5 minutes

• Chromatin Preparation: Sonicate using Bioruptor to achieve 200-500bp fragments

• Pre-clearing: Incubate chromatin with protein A/G beads for 1 hour at 4°C

• Immunoprecipitation: Incubate pre-cleared chromatin with SPAPB24D3.06c antibody overnight at 4°C

• Capture: Add protein A-Sepharose beads for 30 minutes , then wash 3× with wash buffer

• Elution: Release DNA-protein complexes by heating at 65°C for 10 minutes in SDS buffer

• Reverse Crosslinking: Incubate at 65°C overnight

• DNA Purification: Treat with Proteinase K, then purify DNA

• Analysis: Perform qPCR using primers for regions of interest

Critical Considerations:

• Use 5-10μg antibody per IP reaction

• Include IgG control to assess background

• Use antibody against H3 or RNAPII as a positive control

• Compare results between tagged and untagged strains to validate specificity

Recommended Quantification Method:

• For each position, normalize ChIP signal to a reference gene (e.g., act1) and then to histone H3 levels from the same chromatin preparation

What protocols should be followed for using SPAPB24D3.06c antibody in coimmunoprecipitation (Co-IP) studies?

For protein interaction studies using SPAPB24D3.06c antibody in Co-IP:

Cell Preparation and Lysis:

• Grow S. pombe to mid-log phase (~5 × 10^6 cells/ml)

• If studying stress response, treat cells with appropriate stimulus (e.g., 1mM H₂O₂ for 15 minutes)

• Harvest cells and lyse in appropriate buffer (e.g., 50mM HEPES pH 7.5, 140mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate with protease inhibitors)

• Clear lysate by centrifugation (13,000 rpm, 10 minutes, 4°C)

Immunoprecipitation:

• Pre-clear lysate with protein A beads (1 hour, 4°C)

• Incubate pre-cleared lysate with SPAPB24D3.06c antibody (typically 2-5μg) overnight at 4°C

• Add protein A-Sepharose beads and incubate for 2 hours at 4°C

• Wash beads 4× with lysis buffer and 1× with wash buffer

• Elute bound proteins with SDS sample buffer (95°C, 5 minutes)

Analysis of Interacting Partners:

• Separate eluted proteins by SDS-PAGE

• Analyze by Western blot with antibodies against suspected interaction partners

• For unbiased analysis, submit samples for mass spectrometry identification

Validation Approaches:

• Perform reciprocal Co-IP with antibodies against identified partners

• Include negative controls (pre-immune serum, IgG, or unrelated antibody)

• Verify interactions using alternative methods (e.g., yeast two-hybrid or bimolecular fluorescence complementation)

How should researchers interpret and validate SPAPB24D3.06c antibody specificity?

Validating antibody specificity is critical for reliable data interpretation. For SPAPB24D3.06c:

Basic Validation Methods:

• Genetic Validation:

  • Compare Western blot results between wild-type and knockout/deletion strains

  • If deletion is lethal (being an essential gene), use conditional mutants or RNAi-mediated knockdown

• Molecular Validation:

  • Compare detection in strains expressing tagged SPAPB24D3.06c (e.g., HA, FLAG, or myc tag)

  • Use multiple antibodies targeting different epitopes if available

• Biochemical Validation:

  • Pre-absorb antibody with immunizing antigen (supplied with some commercial antibodies)

  • Use the pre-immune serum provided as a negative control

Quantitative Specificity Assessment:

• Signal-to-noise ratio should be >10:1 in Western blots comparing positive and negative samples

• Cross-reactivity should be <10% with unrelated proteins

Advanced Validation Methods:

• IP-Mass Spectrometry:

  • Perform immunoprecipitation followed by mass spectrometry to identify all proteins pulled down

  • Should predominantly identify SPAPB24D3.06c and known interaction partners

• Orthogonal Detection:

  • Compare antibody results with GFP-fluorescence for GFP-tagged SPAPB24D3.06c

  • Compare with RNA-level detection methods (qRT-PCR) for correlation

What are key considerations when analyzing SPAPB24D3.06c expression changes across experimental conditions?

When analyzing SPAPB24D3.06c expression data:

Normalization Strategies:

• For Western blot data:

  • Normalize to total protein load (verified by Ponceau S)

  • Use housekeeping proteins (α-tubulin or Act1) as loading controls

  • Apply densitometry using standard software (ImageJ, ImageQuant TL)

• For qPCR data:

  • Normalize to reference genes stable under your experimental conditions

  • Calculate relative expression using 2^(-ΔΔCt) method

  • Perform technical duplicates or triplicates

Statistical Analysis Framework:

• Perform minimum 3 biological replicates for each condition

• Apply appropriate statistical tests:

  • t-test for comparing two conditions

  • ANOVA for multiple conditions

  • Non-parametric tests if data doesn't follow normal distribution

• Set significance threshold (p<0.05, p<0.01, p<0.001)

Experimental Factors to Consider:

• Cell Cycle Effects: SPAPB24D3.06c expression may vary across cell cycle phases

• Stress Response: Consider whether expression changes represent stress response

  • HDAC activity and antisense transcription can affect gene expression in stress

• Post-translational Modifications: Consider phosphorylation or other modifications

  • Lambda phosphatase treatment can identify phosphorylated forms

Data Visualization:

• Present data with appropriate error bars (SEM for displaying precision of means)

• Include representative blot images alongside quantification

• Use consistent scaling across comparable datasets

How can SPAPB24D3.06c antibody be utilized in chromatin dynamics and transcription research?

The antibody can be leveraged for sophisticated chromatin and transcription studies based on approaches seen in related research:

ChIP-seq Applications:

• Global Binding Profile:

  • Perform ChIP followed by next-generation sequencing to map all genomic binding sites

  • Compare binding profiles under different conditions (e.g., normal vs. stress)

  • Integrate with RNA-seq data to correlate binding with gene expression changes

• Chromatin Association Analysis:

  • Investigate co-occupancy with histone marks (H3K36me3, H3K14ac)

  • Examine relationship with RNA Polymerase II (using antibodies against different phosphorylated forms of RNAPII CTD)

  • Study cell cycle-dependent chromatin association

NET-seq Integration:

• Compare SPAPB24D3.06c binding with native elongating transcript sequencing (NET-seq) data

• Assess correlation with transcriptionally engaged RNA polymerase

• Methodology:

  • Immunoprecipitate using antibody against RNA Pol II (e.g., against Rpb3-flag)

  • Map nascent transcripts

  • Perform differential analysis between wildtype and mutant samples

  • Correlate with SPAPB24D3.06c binding sites

Regulatory Network Analysis:

• Combine with antibodies against other chromatin factors

• Analyze sequential ChIP (Re-ChIP) to identify co-occupied regions

• Integrate with Hi-C or other chromosome conformation data

What experimental approaches can determine if SPAPB24D3.06c is involved in antisense lncRNA-mediated gene regulation?

Based on search results indicating potential involvement in antisense transcription regulation :

Integrated Genomic Analysis:

• RNA-seq in SPAPB24D3.06c mutants:

  • Compare sense and antisense transcription globally

  • Look for genes with altered sense/antisense ratios

  • Focus on genes like ctt1 that show antisense transcription regulation

• ChIP-seq correlation:

  • Map SPAPB24D3.06c binding relative to antisense transcription start sites

  • Correlate with histone modifications associated with transcription initiation/elongation

Mechanistic Investigation:

• RNA Immunoprecipitation (RIP):

  • Use SPAPB24D3.06c antibody to pull down associated RNAs

  • Identify bound lncRNAs through RT-qPCR or sequencing

  • Compare binding to sense vs. antisense transcripts

• Functional Studies:

  • Test effects of SPAPB24D3.06c depletion on antisense transcription

  • Analyze impact on sense transcription of XUT-regulated genes

  • Investigate relationship with exosome components (e.g., Exo2)

Quantitative Analysis Method:

• Perform strand-specific RT-qPCR as described in search result :

  • Use strand-specific primers with actinomycin D (6.25 μg/ml) during reverse transcription

  • Perform qPCR with gene-specific primers

  • Normalize to stable reference RNA (e.g., U3B snoRNA)

  • Compare antisense:sense ratios between wildtype and mutant cells

What are common technical challenges with SPAPB24D3.06c antibody and their solutions?

Western Blot Issues:

ChIP Optimization:

Immunofluorescence Issues:

• Poor signal: Fix cells with methanol , optimize antibody concentration

• Non-specific staining: Include additional blocking agents (normal serum), pre-absorb antibody

• Autofluorescence: Include appropriate controls, use longer wavelength fluorophores

How can researchers optimize SPAPB24D3.06c antibody protocols for challenging applications?

For Low Abundance Detection:

• Signal Amplification:

  • Use high-sensitivity ECL substrates for Western blots

  • Employ tyramide signal amplification for immunohistochemistry

  • Consider biotin-streptavidin detection systems

• Protein Enrichment:

  • Perform subcellular fractionation to concentrate chromatin fraction

  • Use immunoprecipitation prior to Western blot

  • Apply TCA precipitation to concentrate proteins

For Post-translational Modification Studies:

• Modification-Specific Detection:

  • Treat samples with specific enzymes (phosphatases, deglycosylases) to identify modifications

  • Use phos-tag gels to separate phosphorylated forms

  • Compare migration patterns before and after treatment

• Combined Approaches:

  • Perform sequential immunoprecipitation (first with SPAPB24D3.06c antibody, then with modification-specific antibodies)

  • Use 2D gel electrophoresis to separate modified forms

For Interaction Studies:

• Crosslinking Optimization:

  • Test different crosslinkers for protein-protein interactions

  • Optimize crosslinking time and concentration

  • Use protein proximity labeling methods (BioID, APEX)

• Stringency Adjustment:

  • Modify salt concentration in washing buffers to adjust stringency

  • Test different detergent types and concentrations

  • Vary incubation times to optimize signal-to-noise ratio

How might SPAPB24D3.06c antibody be utilized in understanding DNA repair pathways?

The potential relationship between SPAPB24D3.06c and DNA repair can be investigated based on connections to repair pathways seen in search results :

DNA Damage Response Studies:

• Damage-Induced Localization:

  • Track SPAPB24D3.06c localization after treatment with DNA damaging agents using the antibody

  • Compare with localization of known repair factors

  • Analyze recruitment dynamics using time-course experiments

• Pathway Interaction Analysis:

  • Investigate association with base excision repair factors (related to OGG1)

  • Test co-immunoprecipitation with repair machinery components

  • Examine modification status after DNA damage

Methodological Approach:

• Treat cells with oxidative damage agents (H₂O₂, menadione)

• Fix cells at various timepoints

• Perform immunostaining or biochemical fractionation

• Use antibodies against γH2AX as damage markers

• Analyze colocalization with repair factors

Quantitative Assessment:

• Measure SPAPB24D3.06c recruitment to damage sites using ChIP-qPCR at known fragile sites

• Analyze kinetics of association/dissociation following damage

• Compare recruitment in wild-type vs. repair-deficient backgrounds

What new methodologies could incorporate SPAPB24D3.06c antibody for systems biology approaches?

Emerging systems biology applications could include:

Multi-omics Integration:

• Integrative Analysis Pipeline:

  • ChIP-seq with SPAPB24D3.06c antibody → map genomic binding sites

  • RNA-seq → correlate binding with expression changes

  • Proteomics (IP-MS) → identify protein interaction network

  • Integrate all datasets to build comprehensive regulatory model

• Network Construction:

  • Use antibody in different genetic backgrounds to build conditional interaction networks

  • Apply machine learning to predict functional relationships

  • Validate key nodes through targeted experiments

Advanced Imaging Applications:

• Super-resolution Microscopy:

  • Use fluorescently labeled SPAPB24D3.06c antibody for STORM or PALM imaging

  • Map nanoscale distribution on chromatin

  • Analyze co-localization with other factors at single-molecule resolution

• Live-cell Dynamics:

  • Combine with genetically encoded tags for comparative studies

  • Analyze dynamics using FRAP or single-particle tracking

  • Correlate with cell cycle or stress response markers

Computational Modeling Integration:

• Use antibody-generated data to parameterize models of chromatin function

• Validate model predictions through targeted antibody-based experiments

• Develop predictive frameworks for SPAPB24D3.06c function under various conditions