SPAC4H3.03c Antibody

What is SPAC4H3.03c and what is its function in Schizosaccharomyces pombe?

SPAC4H3.03c is a gene encoding a cell wall-remodeling enzyme in the fission yeast Schizosaccharomyces pombe. Research indicates that this protein plays a significant role in cell wall biosynthesis and restructuring. Specifically, it has been identified as being transcriptionally regulated during flocculation processes alongside other cell wall-remodeling genes including gas2+ and psu1+ . The protein appears to be involved in cell wall integrity pathways and may contribute to β-glucan synthesis or modification. Current research suggests it works in concert with other cell wall modification enzymes like Gas2p, which is involved in the β-1,3-glucanosyl-transferase GH72 family that plays a crucial role in maintaining proper cell wall architecture .

How can I confirm the specificity of my SPAC4H3.03c antibody?

Confirming antibody specificity is critical due to documented issues with antibody reproducibility in research. A multi-faceted approach is recommended:

• Genetic validation: Test the antibody in wild-type strains versus SPAC4H3.03c deletion mutants

• Western blot analysis: Look for a single band of the expected molecular weight

• Epitope blocking: Pre-incubate the antibody with the immunizing peptide to demonstrate signal loss

• Orthogonal methods: Compare protein localization using tagged versions of SPAC4H3.03c

• Mass spectrometry validation: Perform immunoprecipitation followed by mass spectrometry to confirm antibody captures the intended target

Research has shown that an alarming number of researchers have encountered irreproducibility in antibody-based experiments, often due to batch variability even when antibodies are sold under the same catalog number .

What experimental techniques can be used to study SPAC4H3.03c localization?

Several techniques can be employed to study the localization of SPAC4H3.03c:

For optimal results with immunolocalization, researchers often use a spheroblasting approach followed by proteinase K protection assays to distinguish between cytoplasmic, membrane-spanning, and cell wall-associated fractions of the protein .

What is the best way to store and handle SPAC4H3.03c antibody to maintain its activity?

To maintain optimal activity:

• Store antibody aliquots at -20°C or -80°C to prevent freeze-thaw cycles

• Add glycerol (final concentration 50%) for long-term storage

• For working stocks, store at 4°C with 0.02% sodium azide

• Avoid repeated freeze-thaw cycles which can lead to antibody degradation and aggregation

• Test antibody activity periodically against known positive controls

• Keep detailed records of lot numbers, as batch variability has been documented as a major cause of irreproducibility

How is SPAC4H3.03c transcriptionally regulated during different physiological conditions?

SPAC4H3.03c exhibits specific transcriptional regulation patterns that researchers should consider:

• Flocculation conditions: SPAC4H3.03c is transcriptionally activated by transcription factors Adn2 and Adn3, which contribute to mild flocculation through cell wall remodeling rather than through classical flocculin pathways .

• Stress responses: Though not directly documented for SPAC4H3.03c, S. pombe cell wall genes often show regulation during stress conditions similar to those seen in histone deacetylase (HDAC) mutants. Class I HDACs like Hos2 and Clr6, and Class II HDACs like Clr3 have been shown to affect gene expression of cell wall components .

• Cell cycle dependency: Many S. pombe genes involved in cell wall synthesis show periodic expression, particularly during cytokinesis when new cell wall material is being deposited at the septum .

• Nutritional conditions: Genes involved in cell wall remodeling often respond to carbon source changes, with glycerol and ethanol serving as common inducers of cell wall modifications .

To study these regulations, researchers should employ techniques like RT-qPCR and chromatin immunoprecipitation to identify transcription factors binding to the SPAC4H3.03c promoter region.

What is the relationship between SPAC4H3.03c and DNA damage response pathways in S. pombe?

While SPAC4H3.03c itself has not been directly implicated in DNA damage response, several relations can be investigated:

• S. pombe SPAC4H3.05 (Srs2), a helicase involved in error-free post-replication repair (PRR), shows genetic interactions with chromatin assembly factors . This suggests a potential connection between cell wall integrity and DNA damage response networks.

• Chromatin assembly factor 1 (CAF-1) has been shown to promote Rad51-dependent homologous recombination . Since cell wall stress often affects nuclear processes, SPAC4H3.03c may indirectly influence DNA damage response through cell integrity signaling.

• To investigate potential connections, researchers should perform epistasis analysis between SPAC4H3.03c deletion mutants and known DNA repair pathway components, using DNA-damaging agents like methyl methanesulfonate (MMS) or UV radiation.

• Co-immunoprecipitation experiments with SPAC4H3.03c antibody could identify potential protein interactions with DNA repair machinery components.

How does SPAC4H3.03c interact with other components of cell wall biosynthesis?

SPAC4H3.03c functions within a complex network of cell wall biosynthesis components:

• Gas2 pathway: SPAC4H3.03c likely works in concert with Gas2p, a β-1,3-glucanosyl-transferase of the GH72 family responsible for cross-linking nascent β-1,3-glucan chains. In S. pombe, this pathway is critical for proper septum formation during cytokinesis .

• Glucan synthases: The S. pombe Bgs1-4 proteins are responsible for β-1,3-glucan synthesis, which forms the major structural component of the cell wall. SPAC4H3.03c may modify these polymers after their initial synthesis .

• O-mannosylation: Cell wall proteins in S. pombe are often O-mannosylated by protein O-mannosyl transferases. The relation between SPAC4H3.03c and this modification pathway could be investigated through glycoprotein-specific staining methods like PAS-Silver staining .

To study these interactions, researchers should employ:

• Co-immunoprecipitation using SPAC4H3.03c antibody followed by mass spectrometry

• Bimolecular Fluorescence Complementation (BiFC) to visualize protein interactions in vivo

• Genetic interaction studies using deletion or conditional mutants of cell wall synthesis genes

What are the best controls when using SPAC4H3.03c antibody in chromatin immunoprecipitation studies?

For reliable ChIP experiments with SPAC4H3.03c antibody:

• Negative controls:

  • IgG control matching the host species of the primary antibody

  • ChIP in SPAC4H3.03c deletion strains to establish background signal

  • Non-target regions (heterochromatic regions are often used)

• Positive controls:

  • ChIP of known chromatin-associated proteins (e.g., histones)

  • Use of epitope-tagged SPAC4H3.03c with commercial tag antibodies

• Input normalization:

  • Always normalize to input chromatin to account for differences in starting material

• Technical considerations:

  • Test multiple antibody concentrations to determine optimal signal-to-noise ratio

  • Include spike-in controls from another species for quantitative normalization

  • Sequence verification of enriched regions to confirm specificity

For S. pombe specifically, researchers have successfully performed ChIP using similar approaches for analyzing histone modifications and chromatin-associated factors .

What are the optimal conditions for using SPAC4H3.03c antibody in Western blotting?

Based on protocols developed for similar S. pombe antibodies:

Researchers should note that some cell wall proteins can be difficult to extract and may require specialized extraction methods involving cell wall digestion with glucanases .

How can I troubleshoot non-specific binding when using SPAC4H3.03c antibody?

Non-specific binding is a common issue that can be addressed systematically:

• Increase stringency of washing:

  • Use higher concentrations of detergent (0.1-0.3% Tween-20)

  • Increase salt concentration (up to 500mM NaCl)

  • Add 0.1% SDS to wash buffers for Western blots

• Optimize blocking:

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

  • Increase blocking time to 2 hours or overnight

  • Add 0.1-1% of host serum to antibody dilutions

• Antibody dilution:

  • Test serial dilutions to identify optimal concentration

  • Pre-absorb antibody with acetone powder from knockout strains

• Sample preparation:

  • Ensure complete reduction of disulfide bonds

  • Consider using fresh samples to avoid degradation products

• Negative controls:

  • Include knockout/deletion strains as negative controls

  • Use pre-immune serum when available

Since batch variability has been identified as a major source of antibody irreproducibility, researchers should maintain detailed records of antibody performance by lot number .

What protein extraction methods are most effective for detecting SPAC4H3.03c?

For optimal extraction of SPAC4H3.03c, consider these protocols adapted for cell wall-associated proteins:

• Standard protocol for cytoplasmic/membrane proteins:

  • Lyse cells with glass beads in buffer containing 50mM Tris pH 7.5, 150mM NaCl, 0.5% NP-40, 1mM EDTA, with protease inhibitors

  • Centrifuge to separate soluble fraction

  • Suitable for detecting cytoplasmic or peripheral membrane portions of SPAC4H3.03c

• Protocol for cell wall-associated proteins:

  • Spheroplast cells using zymolyase or lysing enzymes

  • Extract with hot SDS or alkaline extraction (30mM NaOH)

  • This method releases GPI-anchored and covalently linked cell wall proteins

• Sequential extraction method:

  • Extract with low-strength buffer first (50mM Tris, low salt)

  • Follow with stronger detergent extractions (1% Triton X-100)

  • Finally extract with SDS or alkali

  • This creates fractions enriched for different cellular compartments

• Specialized method for S. pombe cell wall proteins:

  • Include a cell wall biotinylation step to specifically label surface-exposed proteins

  • Perform PAS-Silver staining to detect glycosylated proteins

When working with cell wall proteins, researchers should be aware that standard protein quantification methods may underestimate heavily glycosylated proteins.

How can I optimize SPAC4H3.03c antibody for immunoprecipitation studies?

For successful immunoprecipitation of SPAC4H3.03c:

• Buffer optimization:

  • Test different lysis conditions (RIPA, NP-40, digitonin)

  • Adjust salt concentration (150-500mM NaCl)

  • Consider adding stabilizers like glycerol (5-10%)

• Antibody coupling:

  • Direct coupling to beads (Protein A/G, magnetic) prevents antibody contamination in eluates

  • Cross-linking with DMP or BS3 prevents antibody leaching

  • Use 2-5μg antibody per IP reaction as starting point

• Pre-clearing samples:

  • Pre-clear lysates with beads alone to reduce non-specific binding

  • Include competitor proteins (BSA) in wash buffers

• Controls:

  • Include IgG control matching antibody species

  • Perform IP in deletion strains to confirm specificity

  • Use epitope-tagged SPAC4H3.03c as positive control

• Elution strategies:

  • Peptide elution for preserving protein-protein interactions

  • SDS elution for maximum recovery

For studies of protein interaction networks, researchers should consider tandem affinity purification approaches using both SPAC4H3.03c antibody and antibodies against suspected interaction partners .

How do I interpret conflicting results between different SPAC4H3.03c antibody lots?

Batch variability is a significant problem in antibody research . To address conflicting results:

• Validate each lot independently:

  • Test against known positive and negative controls

  • Perform titration experiments to determine optimal working concentration

  • Compare staining/binding patterns using multiple detection methods

• Document lot-specific characteristics:

  • Keep detailed records of performance by lot number

  • Note differences in background, sensitivity, and specificity

  • Establish lot-specific protocols if necessary

• Perform orthogonal validation:

  • Confirm key results with alternative techniques (e.g., tagged protein)

  • Use genetic approaches (knockouts, siRNA) to complement antibody approaches

  • Consider protein sequencing of immunoprecipitated material

• Scientific publishing considerations:

  • Report antibody lot numbers in publications

  • Clearly describe validation procedures for each lot

  • Acknowledge potential lot-dependent limitations

Researchers should be aware that differences between antibody batches may result from cell-culturing environments or different producing animals, requiring rigorous testing of each new lot .

How can I distinguish between specific SPAC4H3.03c signal and cross-reactivity with related proteins?

To distinguish specific from non-specific signals:

• Genetic approaches:

  • Test antibody in SPAC4H3.03c deletion strains

  • Use strains with varying expression levels (overexpression, repression)

  • Test in related gene deletion backgrounds to check cross-reactivity

• Biochemical approaches:

  • Perform peptide competition assays

  • Use recombinant protein as a standard

  • Employ 2D gel electrophoresis to separate closely related proteins

• Mass spectrometry validation:

  • Analyze immunoprecipitated material by mass spectrometry

  • Identify peptides unique to SPAC4H3.03c versus related proteins

• Epitope mapping:

  • Determine which region of SPAC4H3.03c the antibody recognizes

  • Compare sequence homology with potential cross-reactive proteins

S. pombe has several other cell wall-remodeling enzymes that may share sequence similarity with SPAC4H3.03c, including members of the GH72 family such as Gas1p, Gas2p, Gas4p, and Gas5p .

What factors affect the reproducibility of SPAC4H3.03c detection in different experimental systems?

Several factors impact reproducibility:

• Sample preparation variables:

  • Growth conditions (media, temperature, growth phase)

  • Cell wall composition varies with carbon source and stress conditions

  • Extraction method efficiency varies with cell wall state

• Technical variables:

  • Antibody concentration and incubation conditions

  • Blocking agent compatibility

  • Detection system sensitivity and dynamic range

• Biological variables:

  • Expression level changes with environmental conditions

  • Post-translational modifications affect epitope accessibility

  • Protein localization changes with cell cycle or stress

• Antibody-specific variables:

  • Lot-to-lot variability in specificity and sensitivity

  • Storage conditions and freeze-thaw cycles

  • Cross-reactivity profiles

To enhance reproducibility, researchers should standardize experimental conditions, validate each antibody lot, and include appropriate controls in each experiment. The documented reproducibility crisis in antibody research highlights the importance of these practices .

How should I interpret changes in SPAC4H3.03c localization or expression under different experimental conditions?

Interpreting changes in SPAC4H3.03c requires careful consideration:

• Expression level changes:

  • Normalize to appropriate housekeeping controls

  • Consider whether changes affect total protein or just extractable fraction

  • Verify with orthogonal methods (qRT-PCR, reporter constructs)

• Localization changes:

  • Use multiple markers to confirm subcellular compartments

  • Consider fixation artifacts, especially for cell wall proteins

  • Use live-cell imaging with tagged proteins to confirm antibody-based localization

• Environmental responses:

  • Cell wall remodeling genes often respond to cell wall stress and carbon source

  • SPAC4H3.03c may be regulated alongside other cell wall genes during flocculation

  • Consider regulation by HDAC complexes that affect gene expression

• Technical considerations:

  • Changes in protein extractability can be misinterpreted as expression changes

  • Altered glycosylation may affect antibody recognition

  • Post-translational modifications may create or mask epitopes

When interpreting experimental results, researchers should employ multiple independent techniques and appropriate controls to distinguish true biological changes from technical artifacts.

How can SPAC4H3.03c antibody be used to study cell wall dynamics during the cell cycle?

SPAC4H3.03c antibody can provide valuable insights into cell wall remodeling during cell division:

• Time-course experiments:

  • Synchronize S. pombe cultures (nitrogen starvation, lactose gradient, cdc25 temperature-sensitive mutants)

  • Sample at regular intervals throughout cell cycle

  • Track SPAC4H3.03c localization and abundance by immunofluorescence and Western blotting

• Co-localization studies:

  • Combine SPAC4H3.03c antibody with septum markers (Calcofluor White)

  • Use antibodies against other cell cycle-regulated proteins

  • Correlate with DNA content using DAPI staining

• Live-cell experiments:

  • Compare antibody results with GFP-tagged SPAC4H3.03c

  • Perform time-lapse microscopy to track dynamic changes

  • Correlate with cell cycle progression markers

S. pombe cell wall synthesis proteins like Bgs1-4 show distinct localization patterns during cell division, particularly at the septum during cytokinesis . SPAC4H3.03c likely follows similar patterns if involved in cell wall remodeling during division.

What approaches can be used to study the role of SPAC4H3.03c in cell wall integrity pathways?

To investigate SPAC4H3.03c's role in cell wall integrity:

• Genetic approaches:

  • Create conditional mutants (repressible promoters, temperature-sensitive alleles)

  • Perform epistasis analysis with known cell wall integrity pathway components

  • Screen for synthetic lethality with other cell wall genes

• Physiological testing:

  • Challenge cells with cell wall stressors (Calcofluor White, Congo Red)

  • Test osmotic stability and sensitivity to cell wall-degrading enzymes

  • Examine changes in β-glucan content using specific dyes

• Molecular approaches:

  • Use SPAC4H3.03c antibody to track protein abundance during stress responses

  • Perform ChIP to identify transcription factors regulating SPAC4H3.03c

  • Study protein modifications in response to cell wall stress

• Ultrastructural analysis:

  • Examine cell wall architecture by electron microscopy

  • Compare wild-type and mutant strains

  • Correlate with biochemical composition analysis