SPAC17H9.02 Antibody

What is the SPAC17H9.02 protein and why is it studied?

SPAC17H9.02 (Mtl1) is an essential Mtr4-like RNA helicase in S. pombe that forms part of the nuclear RNA silencing (NURS) complex . It contains conserved domains including DEXDc, HELICc, KOW Mtr4, and DSHCT that are also present in human hMtr4/SKIV2L2 . The protein plays crucial roles in:

• Nuclear RNA processing for heterochromatin assembly

• Association with Red1 protein to form the NURS complex

• Post-transcriptional regulation of meiotic genes

This protein is studied to understand fundamental RNA processing mechanisms and nuclear silencing pathways in eukaryotic cells.

What are the validated applications for the SPAC17H9.02 antibody?

According to the manufacturer's specifications, the SPAC17H9.02 antibody has been tested and validated for:

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Western Blotting (WB) for identification of antigen

While these are the manufacturer-validated applications, researchers should perform their own validation for specific experimental conditions and additional applications such as:

• Immunoprecipitation (IP) for protein complex analysis

• Immunofluorescence for subcellular localization

• Chromatin immunoprecipitation (ChIP) for DNA-protein interaction studies

How should the SPAC17H9.02 antibody be properly stored and handled?

For optimal performance and longevity:

• Store at -20°C or -80°C upon receipt

• Avoid repeated freeze-thaw cycles, which can damage antibody structure and reduce activity

• The antibody is supplied in liquid form with a storage buffer containing:

  • 0.03% Proclin 300 (preservative)

  • 50% Glycerol

  • 0.01M PBS, pH 7.4

When working with the antibody, allow it to equilibrate to room temperature before opening the vial and maintain sterile conditions to prevent contamination.

What controls should be included when using SPAC17H9.02 antibody in Western blotting?

Proper experimental design requires inclusion of appropriate controls:

Positive controls:

• Wild-type S. pombe (strain 972) cell lysates expressing SPAC17H9.02 protein

• The antibody should detect a protein band of approximately 55 kDa in Western blot analysis

Negative controls:

• SPAC17H9.02 knockout strain lysates (when available)

• Isotype control (rabbit IgG) to assess non-specific binding

• Pre-immune serum from the same rabbit used for antibody generation

Additional validation:

• Competition assay with recombinant SPAC17H9.02 protein to confirm specificity

• Load gradient dilutions of sample to assess linear detection range

• Include protein molecular weight markers to confirm target protein size

How can the specificity of SPAC17H9.02 antibody be verified for novel applications?

Establishing antibody specificity is crucial, especially for applications beyond those validated by the manufacturer:

• Gene knockout/knockdown validation:

  • Test antibody on lysates from cells with SPAC17H9.02 gene deletion or knockdown

  • Compare with wild-type cells to confirm loss of signal

• Epitope competition:

  • Pre-incubate antibody with excess purified recombinant SPAC17H9.02 protein

  • Observe elimination or reduction of specific signal

• Cross-reactivity assessment:

  • Test reactivity against related proteins (e.g., other Mtr4-like helicases)

  • Test on lysates from other yeast species or organisms

• Mass spectrometry confirmation:

  • Perform immunoprecipitation followed by mass spectrometry to identify captured proteins

  • Verify that SPAC17H9.02 is the predominant protein detected

As demonstrated in studies of antibody validation, this multi-method approach significantly enhances confidence in antibody specificity .

How can SPAC17H9.02 antibody be used to study the NURS complex in S. pombe?

The NURS complex involves multiple protein components interacting with SPAC17H9.02/Mtl1. To study this complex:

• Co-immunoprecipitation approach:

  • Use SPAC17H9.02 antibody for immunoprecipitation from S. pombe lysates

  • Perform Western blotting for known NURS components:

    • Red1 (the main NURS complex component)

    • Red5 (SPBC337.12) - zinc finger protein

    • Ars2 (SPBC725.08) - zinc finger protein

    • Rmn1 (SPBC902.04) - RNA recognition motif protein

    • Iss10 (SPAC7D4.14c) - serine and proline-rich protein

• Sequential immunoprecipitation:

  • First immunoprecipitate with SPAC17H9.02 antibody

  • Elute complexes and perform a second immunoprecipitation with antibodies against other NURS components

  • This approach identifies subcomplexes containing SPAC17H9.02

• Proximity labeling:

  • Express SPAC17H9.02 fused to a biotin ligase (BioID or TurboID)

  • Identify proximal proteins through streptavidin pulldown and mass spectrometry

  • Compare results with traditional immunoprecipitation using SPAC17H9.02 antibody

Results can be analyzed using quantitative proteomics to determine stoichiometry of the NURS complex components.

What methodological approaches can assess SPAC17H9.02 antibody performance in immunofluorescence studies?

While not explicitly validated for immunofluorescence, researchers may adapt this antibody for localization studies:

• Optimization protocol:

  • Test multiple fixation methods (paraformaldehyde, methanol, acetone)

  • Evaluate various permeabilization reagents (Triton X-100, saponin, digitonin)

  • Try different antibody dilutions (1:100 to 1:1000)

  • Test with and without antigen retrieval methods

  • Include blocking optimization (BSA, normal serum, commercial blockers)

• Validation approach:

  • Co-staining with known nuclear body markers

  • Expression of tagged SPAC17H9.02 (e.g., GFP-tagged) to confirm antibody localization pattern

  • SPAC17H9.02 knockdown to confirm signal specificity

• Expected localization pattern:

  • Nuclear foci or bodies (not colocalizing with nucleolus)

  • Possible co-localization with other NURS complex components

How can SPAC17H9.02 antibody be used to investigate its role in RNA processing and heterochromatin formation?

SPAC17H9.02/Mtl1 is involved in nuclear RNA processing linked to heterochromatin assembly. Advanced research approaches include:

• ChIP-sequencing approach:

  • Crosslink protein-DNA complexes in vivo

  • Immunoprecipitate with SPAC17H9.02 antibody

  • Sequence associated DNA to identify genomic binding sites

  • Compare with H3K9me enrichment patterns to correlate with heterochromatin regions

• RIP-sequencing (RNA immunoprecipitation):

  • Crosslink protein-RNA complexes

  • Immunoprecipitate with SPAC17H9.02 antibody

  • Sequence associated RNAs to identify targets

  • Expected targets may include meiotic gene transcripts that are normally silenced

• Integrative analysis:

  • Compare datasets from SPAC17H9.02 RIP-seq with:

    • Red1 RIP-seq data

    • Exosome mutant expression data

    • H3K9me ChIP-seq data

  • Identify overlapping targets to map the complete silencing pathway

What approaches can resolve non-specific binding issues with SPAC17H9.02 antibody in Western blotting?

Non-specific binding is a common challenge with polyclonal antibodies. Address this through:

• Optimization strategies:

  • Increase blocking stringency (5% BSA or milk, overnight at 4°C)

  • Add 0.1-0.5% Tween-20 to wash buffers

  • Use lower antibody concentration (1:1000 to 1:5000 dilution)

  • Include 0.1% SDS in antibody diluent to reduce hydrophobic interactions

  • Try alternative blocking agents (casein, commercial blockers)

• Sample preparation adjustments:

  • Include protease inhibitors in lysis buffer to prevent degradation

  • Optimize protein loading (10-30 μg/lane)

  • Consider membrane type (PVDF vs. nitrocellulose)

  • Test different transfer conditions

• Advanced techniques:

  • Two-dimensional gel electrophoresis to separate proteins by both isoelectric point and molecular weight

  • Pre-adsorb antibody with lysates from SPAC17H9.02 knockout cells

  • Affinity purification of the antibody against recombinant SPAC17H9.02 protein

How should experimental conditions be modified when working with SPAC17H9.02 mutants or knockout models?

When studying SPAC17H9.02 function through genetic manipulation:

• For conditional mutants:

  • Carefully time sample collection after gene repression

  • Include time-course analysis to capture early effects before lethality

  • Monitor protein levels by Western blot to confirm depletion kinetics

  • Consider using temperature-sensitive alleles with sampling at permissive and restrictive temperatures

• For gene knockout approaches:

  • Since SPAC17H9.02 is essential, use heterozygous diploid strains or auxin-inducible degron systems

  • Implement tetracycline-regulated systems for controlled expression

  • Use complementation with wild-type gene to confirm phenotypes are specifically due to SPAC17H9.02 loss

• For domain-specific mutants:

  • Design mutations in specific domains (DEXDc, HELICc, KOW, DSHCT)

  • Verify protein expression levels are comparable to wild-type

  • Confirm antibody epitope is not affected by the mutations

What approaches can address contradictory findings when comparing SPAC17H9.02 antibody results with tagged protein experiments?

Discrepancies between antibody-based detection and tagged protein approaches require systematic investigation:

• Potential causes of discrepancies:

  • Tag interference with protein function or localization

  • Antibody epitope masking in certain protein complexes

  • Overexpression artifacts with tagged constructs

  • Cell fixation affecting epitope accessibility

• Resolution approach:

  • Compare multiple tagging strategies (N-terminal vs. C-terminal tags)

  • Use different tag types (FLAG, HA, GFP) and sizes

  • Express tagged protein at endogenous levels

  • Perform reciprocal co-immunoprecipitation with antibodies against both SPAC17H9.02 and the tag

  • Use proximity labeling approaches as a third independent method

• Reporting guidelines:

  • Document all discrepancies transparently in publications

  • Provide detailed methodological descriptions for both approaches

  • Consider biological explanations for differences (e.g., subcellular compartmentalization, post-translational modifications)

How can phosphorylation status of SPAC17H9.02 be investigated using this antibody?

SPAC17H9.02 undergoes phosphorylation at multiple sites including S14, S17, S20, S21, S41, S55, and S57 . To study these modifications:

• Phosphorylation-specific analysis:

  • Treat samples with/without phosphatase before Western blotting

  • Look for mobility shifts indicating phosphorylation

  • Use Phos-tag SDS-PAGE to enhance separation of phosphorylated forms

  • Compare detection between total SPAC17H9.02 antibody and phospho-specific antibodies (if available)

• Enrichment approaches:

  • Immunoprecipitate SPAC17H9.02 using the antibody

  • Perform phosphoproteomic analysis by mass spectrometry

  • Compare phosphorylation patterns under different conditions:

  Condition	Expected Phosphorylation Pattern
  Vegetative growth	Baseline phosphorylation at S14, S17, S20, S21
  Nutrient starvation	Potential changes in S41, S55, S57 phosphorylation
  Meiotic induction	Altered phosphorylation pattern

• Functional analysis:

  • Create phospho-mimetic (S→D/E) and phospho-dead (S→A) mutants

  • Compare their phenotypes to wild-type

  • Assess how these mutations affect detection by the SPAC17H9.02 antibody

What considerations are important when interpreting SPAC17H9.02 antibody signal in relation to protein complex formation?

SPAC17H9.02/Mtl1 functions within the NURS complex, requiring careful interpretation of antibody signal:

• Complex assembly analysis:

  • Use native PAGE or blue native PAGE to preserve protein complexes

  • Compare signal patterns between denaturing (SDS-PAGE) and native conditions

  • Perform size exclusion chromatography followed by Western blotting to identify complex size

• Epitope accessibility considerations:

  • The antibody epitope may be masked in certain protein-protein interactions

  • Try multiple antibody concentrations and incubation conditions

  • Consider mild detergents to partially expose epitopes without fully disrupting complexes

• Comparative analysis with other NURS components:

  • Create a detection profile using antibodies against multiple NURS components

  • Compare relative abundances in different cellular fractions

  • Expected patterns based on published literature:

  Protein	Expected Co-detection with SPAC17H9.02	Reference
  Red1	Strong co-detection in nuclear fraction
  Red5	Co-detection in nuclear bodies
  Rmn1	Variable co-detection
  Ars2	Co-detection in nuclear fraction

How should researchers integrate antibody-based data with sequencing and functional genomics data?

Modern research requires integration of multiple data types:

• Integrative data analysis approaches:

  • Compare SPAC17H9.02 antibody ChIP-seq with RNA-seq from SPAC17H9.02 mutants

  • Correlate protein levels (Western blot) with functional outcomes

  • Use network analysis to place SPAC17H9.02 in broader cellular pathways

• Multi-omics integration framework:

  • Start with antibody-based protein localization and interaction data

  • Layer with transcriptome changes in mutants

  • Add epigenomic data (H3K9me ChIP-seq) to identify affected genomic regions

  • Incorporate metabolomic changes for comprehensive phenotypic assessment

• Validation through genetic complementation:

  • Express wild-type or mutant SPAC17H9.02 in depletion backgrounds

  • Use the antibody to confirm expression levels

  • Correlate protein levels with rescue of molecular and cellular phenotypes

Can SPAC17H9.02 antibody be adapted for single-cell protein analysis techniques?

While challenging, adapting this antibody for single-cell applications is possible:

• Technical adaptations for single-cell studies:

  • Mass cytometry (CyTOF) using metal-conjugated SPAC17H9.02 antibody

  • Single-cell Western blotting with microfluidic devices

  • Imaging mass cytometry to preserve spatial information

  • Flow cytometry with fluorophore-conjugated antibody (requires permeabilization optimization)

• Validation strategy for single-cell applications:

  • Compare population-averaged results with single-cell distributions

  • Use SPAC17H9.02-GFP fusion as reference for antibody signal calibration

  • Include spike-in controls of cells with known expression levels

• Expected cellular heterogeneity:

  • Cell cycle-dependent expression patterns

  • Potential variability in nuclear body formation

  • Correlation with other heterochromatin components at single-cell level

What methodological approach would enable investigation of SPAC17H9.02 dynamics during cell cycle progression?

To study dynamic changes in SPAC17H9.02 throughout the cell cycle:

• Synchronization and time-course analysis:

  • Synchronize S. pombe cells using established methods (nitrogen starvation, hydroxyurea block, cdc25 temperature-sensitive mutants)

  • Collect samples at defined intervals (every 15-30 minutes)

  • Perform Western blotting with SPAC17H9.02 antibody to track protein levels

  • Co-stain with cell cycle markers to confirm synchronization

• Live-cell imaging adaptation:

  • Use indirect immunofluorescence at fixed timepoints

  • Compare with live-cell imaging of SPAC17H9.02-GFP fusion

  • Track nuclear body formation and dynamics throughout cell cycle

• Quantification approach:

  • Measure SPAC17H9.02 protein levels relative to housekeeping controls

  • Quantify nuclear body number, size, and intensity

  • Correlate with DNA content and septation index

Expected patterns may include changes in nuclear localization or complex formation at specific cell cycle phases, particularly during S-phase when heterochromatin is established.

How does SPAC17H9.02 antibody performance compare with antibodies targeting other nuclear RNA processing factors?

Comparing antibody performance requires systematic analysis:

• Side-by-side comparison framework:

  • Test SPAC17H9.02 antibody alongside antibodies against other NURS components (Red1, Red5)

  • Include antibodies against related RNA helicases (Mtr4)

  • Use identical experimental conditions for fair comparison

  • Assess specificity, sensitivity, and reproducibility

• Performance metrics to evaluate:

  • Signal-to-noise ratio in Western blotting

  • Specific band intensity vs. background

  • Immunoprecipitation efficiency (% of input recovered)

  • Cross-reactivity profile

• Benchmark with human ortholog antibodies:

  • Compare with antibodies against human hMtr4/SKIV2L2

  • Assess conservation of epitopes and binding patterns

  • Evaluate potential for cross-species reactivity

How can SPAC17H9.02 antibody be used in crosslinking and high-throughput approaches?

Modern genomics and proteomics methods can be adapted:

• CLIP-seq (Crosslinking and Immunoprecipitation) approach:

  • UV crosslink RNA-protein complexes in vivo

  • Immunoprecipitate using SPAC17H9.02 antibody

  • Sequence associated RNAs to identify direct binding targets

  • Expected targets may include meiotic transcripts and cryptic unstable transcripts

• ChIP-exo or ChIP-nexus adaptation:

  • Standard ChIP with SPAC17H9.02 antibody

  • Include exonuclease digestion step

  • Map protein-DNA interactions at near-nucleotide resolution

  • Focus analysis on heterochromatic regions and Heterochromatin Islands (HOODs)

• Proximity-dependent biotinylation:

  • Express SPAC17H9.02 fused to BioID or TurboID

  • Identify proximal proteins through streptavidin pulldown

  • Validate interactions using the SPAC17H9.02 antibody in reciprocal co-IPs

These approaches can map the RNA and protein interaction networks of SPAC17H9.02 with high resolution.

What considerations are important when comparing results from different batches of SPAC17H9.02 antibody?

Polyclonal antibodies may show batch-to-batch variation:

• Batch comparison protocol:

  • Run side-by-side Western blots with different antibody lots

  • Use identical samples and conditions

  • Quantify band intensities and background

  • Compare specificity profiles including non-specific bands

• Standardization approaches:

  • Normalize to positive control samples included in each experiment

  • Maintain a reference lysate aliquot for batch testing

  • Record lot-specific optimal dilutions and conditions

• Documentation requirements:

  • Maintain detailed records of antibody lot numbers

  • Include lot information in publications

  • Consider validation experiments for each new lot

How might SPAC17H9.02 antibody be adapted for super-resolution microscopy techniques?

Super-resolution microscopy offers new insights into nuclear organization:

• Adaptation for different super-resolution methods:

  • STORM/PALM: Use photoswitchable fluorophore-conjugated secondary antibodies

  • SIM: Standard immunofluorescence protocol with high signal-to-noise ratio

  • STED: Bright and photostable fluorophores with optimized labeling density

• Optimization considerations:

  • Fixation methods that preserve nuclear ultrastructure

  • Minimizing background through stringent blocking and washing

  • Testing multiple antibody concentrations to achieve optimal labeling density

  • Using small tags (e.g., nanobodies) for primary detection to reduce linkage error

• Expected biological insights:

  • Nanoscale organization of NURS complex components

  • Spatial relationship between SPAC17H9.02 and chromatin

  • Dynamic assembly/disassembly of nuclear bodies

What approaches could enhance SPAC17H9.02 antibody specificity for challenging applications?

For applications requiring exceptional specificity:

• Affinity purification strategies:

  • Purify the polyclonal antibody against immobilized recombinant SPAC17H9.02

  • Deplete cross-reactive antibodies using knockout cell lysates

  • Generate monoclonal antibodies through hybridoma or phage display technologies

• Epitope-focused approaches:

  • Identify the specific epitope recognized by the polyclonal antibody

  • Generate synthetic peptide-specific antibodies to unique regions

  • Validate specificity against truncated SPAC17H9.02 constructs

• CRISPR epitope tagging:

  • As an alternative approach, use CRISPR to insert a small epitope tag into the endogenous SPAC17H9.02 gene

  • Use well-characterized tag-specific antibodies (FLAG, HA, V5)

  • Compare results with the original SPAC17H9.02 antibody

How can researchers contribute to improving SPAC17H9.02 antibody validation standards?

The research community can enhance antibody reliability through:

• Comprehensive validation reporting:

  • Document all validation experiments in publications

  • Share detailed protocols including optimization steps

  • Report negative results and limitations

  • Deposit validation data in antibody validation repositories

• Multi-method validation approach:

  • Genetic validation using knockout/knockdown systems

  • Orthogonal detection with different antibodies or tagged proteins

  • Application-specific validation for each experimental technique

  • Independent validation across different laboratories

• Community standards adoption:

  • Follow guidelines from the International Working Group for Antibody Validation

  • Implement the "five pillars" of antibody validation

  • Contribute to community resources for antibody validation