SPAC458.02c Antibody

What is the SPAC458.02c protein in S. pombe and what cellular functions has it been implicated in?

SPAC458.02c (UniProt: Q9P3W6) is a protein expressed in Schizosaccharomyces pombe (fission yeast, strain 972/ATCC 24843). Based on the available research, this protein appears to be involved in cellular processes within S. pombe, though detailed functional characterization is still emerging in the literature. Current evidence suggests potential roles in mating-type switching mechanisms, similar to other proteins in fission yeast that participate in homology-directed recombinational repair pathways . The protein may function in processes related to DNA recombination, potentially involving interactions with heterochromatin regions, though direct experimental confirmation is needed for precise functional annotation.

What are the optimal storage conditions for maintaining SPAC458.02c antibody activity?

For optimal preservation of SPAC458.02c antibody activity, store the antibody at -20°C or -80°C upon receipt . The antibody is supplied in liquid form containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . To maintain antibody integrity:

• Avoid repeated freeze-thaw cycles by aliquoting the antibody upon first thaw

• Store working dilutions at 4°C for up to one week

• Return stock aliquots to -20°C or -80°C promptly after use

• When handling, keep the antibody on ice and minimize exposure to room temperature

• Centrifuge briefly before opening the vial to collect any solution that may be trapped in the cap

Proper storage ensures retention of binding specificity and signal strength in downstream applications such as Western blotting and ELISA.

Which experimental applications has the SPAC458.02c antibody been validated for?

The SPAC458.02c antibody (CSB-PA885817XA01SXV) has been validated specifically for ELISA and Western blot applications . The antibody has been affinity-purified using the recombinant SPAC458.02c protein as the immunogen, which enhances its specificity for the target protein. When designing experiments:

For applications beyond Western blot and ELISA, researchers should perform validation studies to determine antibody compatibility and optimal conditions.

What is the recommended protocol for Western blot analysis using SPAC458.02c antibody?

For optimal Western blot results with SPAC458.02c antibody, follow this research-validated protocol:

• Sample preparation:

  • Extract proteins from S. pombe using a lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and protease inhibitor cocktail

  • Quantify protein concentration using Bradford or BCA assay

• Gel electrophoresis and transfer:

  • Separate 20-50 μg of total protein on 10-12% SDS-PAGE

  • Transfer to PVDF membrane at 100V for 60-90 minutes in Towbin buffer

• Antibody incubation:

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

  • Incubate with SPAC458.02c antibody at 1:1000 dilution in blocking buffer overnight at 4°C

  • Wash 3×10 minutes with TBST

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

  • Wash 3×10 minutes with TBST

• Detection and analysis:

  • Develop using ECL substrate and image using a digital imaging system

  • Expected molecular weight of SPAC458.02c should be confirmed based on amino acid sequence analysis

Include positive and negative controls for proper interpretation, such as lysates from wild-type versus SPAC458.02c knockout strains.

How should researchers optimize immunoprecipitation protocols using SPAC458.02c antibody?

While immunoprecipitation (IP) is not explicitly listed among the validated applications for this antibody , researchers may optimize IP protocols as follows:

• Pre-clearing and antibody binding:

  • Pre-clear 500-1000 μg protein lysate with 20 μl Protein A/G beads for 1 hour at 4°C

  • Incubate pre-cleared lysate with 2-5 μg SPAC458.02c antibody overnight at 4°C with gentle rotation

  • Add 30 μl of fresh Protein A/G beads and incubate for 2-4 hours at 4°C

• Washing and elution:

  • Wash beads 4-5 times with IP buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% NP-40)

  • Elute proteins by boiling in 2× Laemmli buffer for 5 minutes at 95°C

• Validation approaches:

  • Perform parallel IPs with pre-immune serum as negative control

  • Include a no-antibody control

  • Verify specificity by Western blot of IP products

  • Consider crosslinking the antibody to beads to prevent antibody co-elution

• Optimization parameters:

  • Test different antibody concentrations (1-10 μg per IP)

  • Vary incubation times (4-16 hours)

  • Test different detergent concentrations in wash buffers

An appropriate positive control would be a known interaction partner of SPAC458.02c, while a negative control could be an unrelated protein of similar abundance.

How might the functional relationship between SPAC458.02c and mating-type switching in S. pombe be investigated?

To investigate potential roles of SPAC458.02c in mating-type switching in S. pombe, researchers should consider a multi-faceted approach:

• Genetic analysis:

  • Generate SPAC458.02c deletion mutants and analyze mating-type switching efficiency

  • Examine genetic interactions with known mating-type switching factors such as Swi2, Swi5, and Swi6

  • Create point mutations in potential functional domains and assess phenotypic outcomes

• Localization studies:

  • Use SPAC458.02c antibody for immunofluorescence to determine subcellular localization

  • Examine co-localization with mating-type loci using fluorescence in situ hybridization (FISH)

  • Investigate dynamics during the cell cycle and mating process

• Biochemical approaches:

  • Perform chromatin immunoprecipitation (ChIP) to determine if SPAC458.02c associates with mating-type loci

  • Identify protein interaction partners using immunoprecipitation followed by mass spectrometry

  • Analyze potential DNA-binding properties, especially if the protein contains motifs similar to the AT-hooks found in Swi2

• Functional assays:

  • Assess the impact of SPAC458.02c on recombination efficiency at mating-type loci

  • Examine its potential involvement in heterochromatin formation or maintenance

  • Analyze whether it influences donor preference during mating-type switching

If SPAC458.02c functions similarly to other switching factors like Swi2, researchers might investigate whether it contains functional domains such as HP1-binding sites or DNA-binding motifs that could mediate interactions with specific genomic regions .

What controls are essential when performing ChIP experiments using SPAC458.02c antibody?

When designing ChIP experiments with SPAC458.02c antibody, include these essential controls:

• Input control:

  • Reserve 5-10% of chromatin prior to immunoprecipitation

  • Use to normalize ChIP signals and account for differences in starting material

• Negative controls:

  • IgG control: Perform parallel ChIP with non-specific rabbit IgG

  • No-antibody control: Process samples without adding primary antibody

  • Negative genomic region: Amplify a genomic region not expected to be bound by SPAC458.02c

• Positive controls:

  • If known binding sites exist, include primers for these regions

  • If SPAC458.02c functions like Swi2, consider examining recombination enhancer regions (SRE2/SRE3)

• Biological controls:

  • SPAC458.02c deletion strain: Should show no enrichment in ChIP

  • Strains with mutations in interacting partners

• Technical validation:

  • Perform sequential ChIP (re-ChIP) to confirm co-occupancy with known interacting proteins

  • Validate enriched regions by independent methods (e.g., EMSA if DNA binding is suspected)

These controls help distinguish genuine chromatin associations from technical artifacts and provide confidence in ChIP results.

What are common causes of non-specific bands in Western blots using SPAC458.02c antibody and how can they be mitigated?

Non-specific bands in Western blots with SPAC458.02c antibody can arise from several sources. Here are common causes and mitigation strategies:

• Cross-reactivity with similar epitopes:

  • Increase blocking stringency (try 5% BSA instead of milk)

  • Optimize antibody dilution (typically 1:1000-1:2000)

  • Use PVDF membranes which may provide lower background than nitrocellulose

  • Consider peptide competition assays to confirm specificity

• Protein degradation products:

  • Use fresh samples and maintain cold chain

  • Add protease inhibitors to lysis buffer

  • Reduce sample processing time

  • Include EDTA (1-5 mM) in lysis buffer

• Insufficient blocking:

  • Extend blocking time to 2 hours at room temperature

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

  • Use 0.1% Tween-20 in wash and antibody dilution buffers

• Antibody concentration issues:

  • Perform titration experiments to determine optimal concentration

  • Consider increasing wash steps (5×10 minutes)

  • Dilute antibody in fresh blocking buffer

• Secondary antibody problems:

  • Ensure secondary antibody is specific to rabbit IgG

  • Test secondary antibody alone (without primary) to check for direct binding

  • Pre-adsorb secondary antibody if necessary

The polyclonal nature of this SPAC458.02c antibody means some batch-to-batch variation may occur. When troubleshooting, always include appropriate positive and negative controls to distinguish specific from non-specific signals.

How should discrepancies between antibody-based detection and transcript levels of SPAC458.02c be interpreted?

Discrepancies between protein levels (detected by antibody) and transcript levels (measured by RT-qPCR or RNA-seq) of SPAC458.02c require careful interpretation:

• Biological explanations:

  • Post-transcriptional regulation: miRNAs or RNA-binding proteins may affect translation efficiency

  • Protein stability differences: Variations in protein half-life due to post-translational modifications

  • Temporal dynamics: Time lag between transcription and translation

  • Cell cycle effects: Expression may vary throughout cell cycle phases

• Technical considerations:

  • Antibody sensitivity: Detection threshold may differ from RNA methods

  • Antibody specificity: Cross-reactivity with related proteins

  • RNA extraction efficiency: Certain transcripts may be lost during isolation

  • Primer efficiency in qPCR: Suboptimal primers may underestimate transcript levels

• Validation approaches:

  • Perform time-course experiments to capture dynamic relationships

  • Use multiple antibodies targeting different epitopes

  • Compare results with tagged versions of SPAC458.02c

  • Employ ribosome profiling to assess translation efficiency

• Quantitative analysis:

  • Plot protein vs. RNA levels across multiple conditions

  • Calculate Pearson correlation coefficients

  • Apply statistical methods that account for technical variability

When interpreting such discrepancies, consider that post-transcriptional and post-translational regulation are normal biological phenomena. The absence of correlation does not necessarily indicate technical issues but may reflect genuine regulatory mechanisms.

How might SPAC458.02c function relate to heterochromatin organization in S. pombe?

Based on the mechanisms observed with related proteins in S. pombe, SPAC458.02c might participate in heterochromatin organization through several potential mechanisms:

• Chromatin remodeling interactions:

  • Similar to Swi2, SPAC458.02c might interact with chromatin-associated proteins like Swi6 (HP1 homolog)

  • The protein could potentially contain functional domains that mediate interactions with heterochromatin components

  • It may participate in complexes that regulate chromatin accessibility at specific genomic regions

• DNA binding capabilities:

  • If SPAC458.02c contains DNA-binding motifs similar to the AT-hooks found in Swi2 , it could directly bind to specific sequences

  • Such binding could facilitate recruitment of chromatin modifiers or recombination machinery

  • Sequence-specific DNA binding might be important for targeted chromatin reorganization

• Recombination and repair processes:

  • SPAC458.02c might function in homology-directed recombination pathways

  • It could potentially interact with recombination-associated proteins like Rad51

  • Such interactions might facilitate specific recombination events within heterochromatic regions

• Cell-type specific regulation:

  • The protein might show cell-type specific localization patterns

  • It could participate in determining donor preference during recombination events

  • Such specificity might be regulated through post-translational modifications

To investigate these possibilities, researchers could perform ChIP-seq to identify genomic binding sites, analyze the protein's domain structure for functional motifs, and examine genetic interactions with known heterochromatin factors and recombination proteins.

What advanced techniques could enhance detection sensitivity when working with low-abundance SPAC458.02c protein?

For improved detection of low-abundance SPAC458.02c protein, consider these advanced techniques:

• Enhanced immunoprecipitation approaches:

  • Tandem affinity purification using dual tags

  • Proximity-dependent biotin identification (BioID) to capture transient interactions

  • Cross-linking immunoprecipitation (CLIP) for enhanced stability during isolation

• Signal amplification in Western blotting:

  • Tyramide signal amplification (TSA) - can increase sensitivity 10-50 fold

  • Poly-HRP conjugated secondary antibodies

  • Chemiluminescent substrates optimized for ultra-sensitive detection

  • Digital accumulation of signal using extended exposure imaging

• Advanced microscopy techniques:

  • Stochastic optical reconstruction microscopy (STORM)

  • Photoactivated localization microscopy (PALM)

  • Expansion microscopy for physical magnification of samples

  • Lattice light-sheet microscopy for reduced phototoxicity during live imaging

• Mass spectrometry approaches:

  • Selected reaction monitoring (SRM) or multiple reaction monitoring (MRM)

  • Parallel reaction monitoring (PRM) for targeted detection

  • TMT or iTRAQ labeling for quantitative comparisons

  • AQUA peptides as internal standards for absolute quantification

• Protein enrichment strategies:

  • Subcellular fractionation to concentrate proteins from relevant compartments

  • Phosphorylation-specific enrichment if SPAC458.02c is phosphorylated

  • Expression of tagged protein in relevant genetic backgrounds for easier detection

Each of these approaches has specific advantages and limitations that should be considered based on your experimental question, available equipment, and sample constraints.