SPAC20H4.09 Antibody

What is SPAC20H4.09 and why is it important in research?

SPAC20H4.09 is an uncharacterized RNA helicase in Schizosaccharomyces pombe that shares similarities with human DHX35. It has generated significant research interest due to its potential role in RNA splicing. Previous studies have shown that both SPAC20H4.09 and Gpl1 (a G-patch domain-containing protein) co-purify with splicing factors, and the SPAC20H4.09Δ mutant shows genetic interactions with mutants defective in splicing. The SPAC20H4.09 antibody is a crucial tool for investigating this protein's function and interactions in molecular biology research, particularly in understanding RNA processing mechanisms .

What are the specifications of commercially available SPAC20H4.09 antibodies?

The SPAC20H4.09 antibody (e.g., CSB-PA864047XA01SXV) is a rabbit polyclonal antibody raised against recombinant Schizosaccharomyces pombe (strain 972 / ATCC 24843) SPAC20H4.09 protein. It is supplied as a liquid in storage buffer containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4. The antibody has been tested for ELISA and Western Blot applications and is purified using antigen affinity methods. It is intended for research use only and should be stored at -20°C or -80°C, avoiding repeated freeze-thaw cycles .

How should I optimize Western blot conditions when using SPAC20H4.09 antibody?

For optimal Western blot results with SPAC20H4.09 antibody:

• Sample preparation: Prepare protein lysates from S. pombe using standard protocols such as those employed in purifying TAP-tagged proteins .

• Gel conditions: Use SDS-PAGE with the appropriate percentage acrylamide gel based on SPAC20H4.09's molecular weight.

• Dilution optimization: Start with a 1:1000 dilution and adjust based on signal intensity.

• Blocking: Use 2-5% BSA or non-fat milk in TBS-T for blocking.

• Incubation time: Incubate with primary antibody overnight at 4°C for best results.

• Detection system: Select an appropriate HRP-conjugated secondary antibody (anti-rabbit IgG) and use enhanced chemiluminescence for visualization.

• Controls: Always include positive controls (purified SPAC20H4.09 protein) and negative controls (lysates from SPAC20H4.09Δ strains) to verify specificity.

These conditions are based on general antibody application principles and should be further optimized for your specific experimental setup .

What immunoprecipitation protocols work best with SPAC20H4.09 antibody?

For effective immunoprecipitation using SPAC20H4.09 antibody, follow this methodology:

• Lysate preparation: Prepare S. pombe cell lysates in a buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and protease inhibitors.

• Pre-clearing: Pre-clear the lysate with Protein A/G beads for 1 hour at 4°C.

• Antibody binding: Incubate 2-5 μg of SPAC20H4.09 antibody with pre-cleared lysate overnight at 4°C with gentle rotation.

• Bead capture: Add Protein A/G beads and incubate for 2-4 hours at 4°C.

• Washing: Wash beads 4-5 times with lysis buffer containing reduced detergent.

• Elution: Elute bound proteins by boiling in SDS-PAGE sample buffer or using a mild elution buffer.

• Analysis: Analyze precipitated proteins by Western blot or mass spectrometry.

This protocol is similar to the TAP purification methodology used to study SPAC20H4.09's interactions with splicing factors .

How can I use SPAC20H4.09 antibody to investigate its interaction with the G-patch protein Gpl1?

To investigate the interaction between SPAC20H4.09 and Gpl1, implement the following experimental strategy:

• Co-immunoprecipitation:

  • Perform reciprocal co-IPs using SPAC20H4.09 antibody and Gpl1 antibody

  • Analyze precipitates by Western blot to detect interacting partners

• Proximity ligation assay (PLA):

  • Use SPAC20H4.09 antibody and Gpl1 antibody as primary antibodies

  • Apply PLA protocol to visualize and quantify protein interactions in situ

• Functional validation:

  • Compare RNA splicing efficiency in wild-type, SPAC20H4.09Δ, gpl1Δ, and double mutant strains

  • Use the antibody to analyze SPAC20H4.09 expression levels in these different genetic backgrounds

• Chromatin immunoprecipitation (ChIP):

  • Employ SPAC20H4.09 antibody for ChIP assays to identify RNA targets

  • Compare results with Gpl1 ChIP patterns to identify co-regulated genes

This approach would build upon previous findings suggesting functional interaction between SPAC20H4.09 and Gpl1 in RNA splicing .

What are the best methodologies for validating SPAC20H4.09 antibody specificity in research applications?

To validate SPAC20H4.09 antibody specificity, employ these methodologies:

• Genetic validation:

  • Test the antibody on wild-type vs. SPAC20H4.09Δ mutant samples

  • The signal should be absent or significantly reduced in knockout samples

• Peptide competition assay:

  • Pre-incubate the antibody with excess SPAC20H4.09 recombinant protein

  • Compare with non-competed antibody; specific signals should be blocked

• Orthogonal detection:

  • Compare detection patterns using multiple antibodies targeting different epitopes

  • Use tagged SPAC20H4.09 (e.g., TAP-tag) and detect with tag-specific antibodies

• Mass spectrometry validation:

  • Immunoprecipitate using SPAC20H4.09 antibody

  • Confirm identity of precipitated proteins by mass spectrometry analysis

• Functional validation:

  • Deplete the antibody with recombinant SPAC20H4.09 and test for loss of functional activity

These approaches follow best practices for antibody validation as outlined in current literature on antibody characterization .

What are common issues when using SPAC20H4.09 antibody in immunofluorescence experiments and how can they be resolved?

For best results, optimize antigen retrieval methods and consider using tyramide signal amplification for low-abundance targets. Additionally, proper validation controls, including SPAC20H4.09Δ samples, should always be included .

How can I distinguish between specific and non-specific binding when using SPAC20H4.09 antibody?

To distinguish between specific and non-specific binding when using SPAC20H4.09 antibody:

• Genetic validation: Compare results between wild-type and SPAC20H4.09Δ samples to identify specific signals.

• Competing peptide controls: Pre-incubate the antibody with excess antigen to block specific binding sites.

• Multiple detection methods: Validate findings using orthogonal techniques (e.g., Western blot, IP-MS, immunofluorescence).

• Titration experiments: Perform antibody dilution series to identify the optimal concentration where specific signal is maintained while background is minimized.

• Isotype controls: Use non-specific rabbit IgG at the same concentration to identify non-specific binding.

• Molecular weight verification: In Western blots, specific binding should appear at the predicted molecular weight of SPAC20H4.09.

• Subcellular localization: Compare observed localization patterns with known or predicted localization of SPAC20H4.09.

These methods align with recommendations for antibody validation in the scientific community and help ensure experimental reproducibility .

How should I interpret Western blot results when detecting SPAC20H4.09 in different genetic backgrounds?

When interpreting Western blot results for SPAC20H4.09 across different genetic backgrounds:

• Band size analysis: The primary band should appear at the predicted molecular weight of SPAC20H4.09.

• Quantitative comparison:

  • Normalize SPAC20H4.09 signal to loading controls (e.g., tubulin, actin)

  • Use densitometry software for quantification

  • Apply appropriate statistical tests for comparing expression levels

• Genetic background effects:

  • In ntr1, ntr2, or brr2 mutants, look for changes in SPAC20H4.09 levels that might indicate co-regulation

  • In gpl1Δ strains, assess whether SPAC20H4.09 levels or post-translational modifications are altered

• Post-translational modifications:

  • Be alert for additional bands that might represent modified forms

  • Verify with phosphatase treatment if phosphorylation is suspected

• Interpretation guidelines:

  • Consistent changes across biological replicates are more reliable

  • Consider both statistical significance and biological relevance

  • Validate key findings with additional techniques

This analytical approach will help establish relationships between SPAC20H4.09 and other splicing factors identified in previous studies .

What controls are essential when performing immunoprecipitation experiments with SPAC20H4.09 antibody?

Essential controls for immunoprecipitation experiments with SPAC20H4.09 antibody include:

• Input control:

  • Analyze 5-10% of pre-IP lysate to confirm target protein presence

  • Essential for quantifying IP efficiency

• Negative controls:

  • IgG control: Non-specific IgG from the same species as SPAC20H4.09 antibody

  • Bead-only control: Beads without antibody to identify proteins binding to the matrix

  • Knockout/knockdown control: Lysate from SPAC20H4.09Δ strains

• Reciprocal IP:

  • For interaction studies, perform reverse IP using antibodies against suspected binding partners

• Competitive inhibition control:

  • Pre-incubate antibody with recombinant SPAC20H4.09 before IP

• Denaturing vs. native conditions:

  • Compare results under different lysis conditions to distinguish direct vs. indirect interactions

• Biological replicates:

  • Perform at least three independent experiments to ensure reproducibility

• Validation by mass spectrometry:

  • Confirm identity of co-immunoprecipitated proteins, especially for previously unknown interactions

These controls align with those used in studies of splicing factor interactions in S. pombe and ensure reliable identification of specific SPAC20H4.09 protein interactions .

How can I use SPAC20H4.09 antibody to investigate its potential role in RNA splicing mechanisms?

To investigate SPAC20H4.09's role in RNA splicing using the antibody:

• RNA-Immunoprecipitation (RIP):

  • Use SPAC20H4.09 antibody to precipitate protein-RNA complexes

  • Identify bound RNAs through sequencing or RT-PCR

  • Compare with known splicing regulators like Ntr1, Ntr2, and Brr2

• Spliceosome complex analysis:

  • Perform glycerol gradient fractionation of cell extracts

  • Use SPAC20H4.09 antibody to track its presence in different spliceosome assembly stages

  • Co-immunoprecipitate to identify stage-specific interaction partners

• Functional splicing assays:

  • Deplete SPAC20H4.09 using RNAi or CRISPR techniques

  • Assess splicing efficiency with reporter constructs

  • Use the antibody to confirm depletion efficacy

• In vitro splicing reconstitution:

  • Immunodeplete SPAC20H4.09 from splicing-competent extracts

  • Test splicing activity before and after depletion

  • Rescue activity by adding back purified protein

• Co-localization studies:

  • Perform immunofluorescence with SPAC20H4.09 antibody and known splicing factors

  • Analyze co-localization in nuclear speckles or other splicing-related compartments

This approach builds on findings that SPAC20H4.09 co-purifies with splicing factors and shows genetic interactions with splicing-defective mutants .

What methodologies can be used to study the regulation of SPAC20H4.09 by G-patch proteins like Gpl1?

To study the regulation of SPAC20H4.09 by G-patch proteins like Gpl1:

• In vitro helicase assays:

  • Purify recombinant SPAC20H4.09 protein

  • Measure RNA unwinding activity with and without Gpl1

  • Use the antibody to immunodeplete endogenous SPAC20H4.09 from extracts

• Structure-function analysis:

  • Generate domain deletion mutants of SPAC20H4.09

  • Use the antibody to immunoprecipitate these mutants with Gpl1

  • Map the interaction domains critical for functional association

• RNA-dependent interaction studies:

  • Perform co-IP with SPAC20H4.09 antibody in the presence/absence of RNase

  • Determine if the interaction with Gpl1 is direct or RNA-mediated

• In vivo proximity labeling:

  • Fuse BioID or APEX2 to SPAC20H4.09 or Gpl1

  • Use antibodies to detect biotinylated proteins in different genetic backgrounds

• Single-molecule studies:

  • Use SPAC20H4.09 antibody for single-molecule pull-down (SiMPull) assays

  • Analyze the dynamic association with Gpl1 at the single-molecule level

• Comparative analysis with other G-patch protein-helicase pairs:

  • Use similar approaches to study other helicase-G-patch protein pairs

  • Compare regulatory mechanisms across different RNA processing pathways

This comprehensive approach would elucidate the molecular mechanism by which G-patch proteins like Gpl1 might regulate the SPAC20H4.09 helicase, as has been observed for other RNA helicases involved in RNA processing .

How should I design experiments to study SPAC20H4.09 localization and dynamics during different cell cycle stages?

To study SPAC20H4.09 localization and dynamics throughout the cell cycle:

• Synchronization and time-course analysis:

  • Synchronize S. pombe cells using established methods (e.g., lactose gradient, hydroxyurea block)

  • Collect samples at defined time points after synchronization

  • Perform immunofluorescence using SPAC20H4.09 antibody

  • Co-stain with cell cycle markers and DNA

• Live-cell imaging:

  • Generate GFP or mCherry fusion constructs of SPAC20H4.09

  • Validate fusion protein localization matches antibody staining patterns

  • Perform time-lapse imaging throughout the cell cycle

• Cell cycle-specific protein interactions:

  • Perform immunoprecipitation with SPAC20H4.09 antibody at different cell cycle stages

  • Identify cell cycle-specific interacting partners by mass spectrometry

  • Validate key interactions by co-immunoprecipitation and co-localization

• Quantitative analysis:

  • Measure changes in SPAC20H4.09 levels by Western blot across the cell cycle

  • Quantify subcellular distribution using high-content imaging

  • Apply appropriate statistical analyses to identify significant changes

• Control experiments:

  • Use cell cycle blocking agents to confirm stage-specific patterns

  • Include cell cycle marker proteins as positive controls

  • Employ SPAC20H4.09Δ strains as negative controls

This experimental design will reveal if SPAC20H4.09's function in RNA processing is regulated in a cell cycle-dependent manner, potentially connecting RNA splicing regulation to cell cycle progression .

What techniques can be combined with antibody-based approaches to comprehensively characterize SPAC20H4.09 function in fission yeast?

A comprehensive characterization of SPAC20H4.09 function requires integrating antibody-based approaches with complementary techniques:

• Genomic approaches:

  • ChIP-seq using SPAC20H4.09 antibody to identify genomic binding sites

  • RNA-seq in wild-type vs. SPAC20H4.09Δ strains to identify regulated transcripts

  • CLIP-seq to map direct RNA binding sites

• Proteomic approaches:

  • Immunoprecipitation followed by mass spectrometry to identify protein interactors

  • Proximity labeling (BioID/APEX) to identify the broader interaction network

  • Phosphoproteomics to identify regulatory post-translational modifications

• Genetic approaches:

  • Synthetic genetic array analysis with SPAC20H4.09Δ to identify genetic interactions

  • CRISPR screens to identify genes that buffer or enhance SPAC20H4.09 function

  • Suppressor screens to identify downstream effectors

• Biochemical approaches:

  • In vitro reconstitution of splicing with purified components

  • RNA helicase assays to characterize enzymatic activity

  • Structure determination by cryo-EM or X-ray crystallography

• Cell biological approaches:

  • Immunofluorescence under various stresses to assess dynamic relocalization

  • FRAP (Fluorescence Recovery After Photobleaching) to measure protein dynamics

  • Super-resolution microscopy to precisely localize SPAC20H4.09 within nuclear structures

This multi-disciplinary approach would provide a comprehensive understanding of SPAC20H4.09's role in RNA processing and potential connections to other cellular pathways .