SPAC2H10.01 Antibody

What is SPAC2H10.01 and why is it significant for yeast research?

SPAC2H10.01 is a gene/protein found in Schizosaccharomyces pombe (fission yeast) with the Entrez Gene ID 2541664 and UniProt number O94392 . It is particularly significant for research involving cell wall proteins and protein glycosylation in fission yeast. S. pombe serves as an excellent model organism for studying fundamental eukaryotic cellular processes, including cell cycle regulation, chromosome dynamics, and protein processing. The SPAC2H10.01 protein may be involved in multiple cellular pathways, and studying it contributes to our understanding of conserved cellular mechanisms that may have parallels in higher eukaryotes, including humans.

The specific functions associated with SPAC2H10.01 are still being investigated, but it appears to be related to cell wall integrity and potentially protein processing pathways, making it relevant for researchers studying these cellular aspects. Investigating this protein using specific antibodies allows researchers to track its expression, localization, and modification under various experimental conditions, providing insights into its functional roles within the cell.

What applications is the SPAC2H10.01 antibody validated for?

The SPAC2H10.01 antibody has been primarily validated for enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) applications . For Western blot applications, this antibody can be used to detect the native protein from yeast cell lysates, allowing researchers to monitor protein expression levels under different conditions or in various mutant strains. The antibody's purification via antigen affinity methods enhances its specificity for the target protein, reducing background signals in these applications.

For ELISA applications, the antibody can be used to quantitatively measure SPAC2H10.01 protein levels in yeast samples. This can be particularly useful for high-throughput screening or when analyzing multiple samples simultaneously. While not explicitly validated for other applications in the provided information, researchers might explore its utility in immunoprecipitation (IP) or immunofluorescence (IF) experiments after appropriate validation tests. Any novel application would require thorough optimization and validation to ensure specificity and sensitivity.

How should the SPAC2H10.01 antibody be stored and handled for optimal performance?

The SPAC2H10.01 antibody should be stored at either -20°C or -80°C for long-term preservation of its activity . When working with the antibody, it's recommended to minimize freeze-thaw cycles by aliquoting the stock solution into smaller volumes appropriate for single-use experiments. Repeated freeze-thaw cycles can lead to protein denaturation and subsequent loss of antibody activity or specificity.

For daily handling, the antibody should be kept on ice while in use and returned to appropriate storage temperatures promptly after use. When diluting the antibody for experimental applications, use high-quality buffers free of contaminants. The antibody product comes with both positive control (200μg antigens) and negative control (1ml pre-immune serum) components, which should be stored according to the same conditions as the antibody itself . These controls should be incorporated into experimental design to validate antibody performance and ensure reliable interpretation of results.

What are the optimal conditions for Western blot detection using SPAC2H10.01 antibody?

When designing Western blot experiments with SPAC2H10.01 antibody, several key parameters should be considered for optimal results. First, since this is a polyclonal antibody raised in rabbits , a secondary anti-rabbit IgG antibody conjugated with HRP or another detection system will be required. Based on general practices with similar antibodies, starting with a primary antibody dilution of 1:1000 to 1:5000 is reasonable, though this should be empirically optimized.

Sample preparation is critical for detecting yeast proteins. Effective lysis methods typically involve mechanical disruption (glass beads or sonication) combined with detergent-based buffers containing protease inhibitors to prevent protein degradation. For S. pombe proteins, spheroplasting methods might be necessary to overcome the rigid cell wall structure, as described in protocols for fission yeast . When preparing samples, include appropriate controls: wild-type yeast, yeast strains with SPAC2H10.01 deletion (if available), and the positive control antigen provided with the antibody . For blocking, 5% non-fat dry milk or 3-5% BSA in TBST is typically effective, though optimization may be necessary. Detection sensitivity can be enhanced using chemiluminescent substrates with varying sensitivity levels depending on the expected abundance of the target protein.

How should ELISA protocols be modified for SPAC2H10.01 antibody?

For ELISA applications with SPAC2H10.01 antibody, several considerations can improve experimental outcomes. When coating plates, purified recombinant SPAC2H10.01 protein or yeast cell extracts containing the protein can be used depending on the experimental question. Standard coating concentrations range from 1-10 μg/ml in carbonate buffer (pH 9.6) overnight at 4°C. After coating, blocking with 1-3% BSA in PBS is typically effective to reduce non-specific binding.

The SPAC2H10.01 antibody should be titrated to determine optimal concentration, typically starting with dilutions between 1:500 and 1:5000. The 200μg antigen provided as a positive control can be used to generate a standard curve for quantitative applications. For detection, an anti-rabbit IgG secondary antibody conjugated with HRP followed by TMB substrate provides a sensitive readout system, similar to approaches used with other antibodies . The reaction should be stopped with sulfuric or phosphoric acid solution after appropriate color development, and absorbance read at 450nm. When designing ELISA experiments, consider including wells with pre-immune serum (provided as negative control ) at the same dilution as the primary antibody to assess background signals.

How can cross-reactivity with other yeast proteins be minimized when using this antibody?

Minimizing cross-reactivity is essential for obtaining reliable results with the SPAC2H10.01 antibody. While this antibody is affinity-purified , which enhances its specificity, several strategies can further reduce potential cross-reactivity. First, perform careful titration experiments to determine the minimal effective concentration of the antibody that provides specific signal with minimal background. Higher antibody concentrations can sometimes increase non-specific binding.

Including additional blocking agents such as 0.1-0.5% Tween-20 in washing and antibody dilution buffers can help reduce non-specific interactions. For particularly challenging samples, pre-absorption of the antibody with yeast lysates lacking the target protein (SPAC2H10.01 knockout strains if available) can remove antibodies that might cross-react with other yeast proteins. When analyzing complex samples, consider using SPAC2H10.01 knockout yeast as a negative control to identify any bands or signals that might represent cross-reactive species.

For critical applications requiring absolute specificity, epitope competition assays can be performed by pre-incubating the antibody with excess purified recombinant SPAC2H10.01 protein before adding to the experimental sample. This should abolish specific signals while leaving any non-specific interactions unchanged, allowing for their identification and mitigation in subsequent experiments.

What are common issues when detecting SPAC2H10.01 in yeast samples and how to resolve them?

Several challenges may arise when detecting SPAC2H10.01 in yeast samples. One common issue is insufficient protein extraction due to the robust cell wall of S. pombe. To address this, effective cell lysis protocols should be employed, such as mechanical disruption with glass beads combined with enzymatic pre-treatment to weaken the cell wall structure. Spheroblasting protocols specifically designed for S. pombe can significantly improve protein extraction efficiency .

Another frequent problem is high background signal in Western blots. This can be addressed by increasing blocking time or concentration (using 5% milk or BSA), adding 0.1-0.5% Tween-20 to washing buffers, and increasing the number and duration of wash steps. If background persists, further diluting the primary antibody or pre-absorbing it with non-specific proteins can help. Additionally, weak or absent signal may occur if the protein is present at low abundance. In such cases, enrichment strategies such as immunoprecipitation prior to Western blotting, or using more sensitive detection reagents like enhanced chemiluminescence substrates, can improve detection.

Post-translational modifications may affect antibody recognition, particularly if they occur within the epitope region. If the protein is suspected to be heavily glycosylated or modified in other ways, treatment with appropriate enzymes (like EndoH for N-glycosylation ) prior to SDS-PAGE can help normalize the protein for better detection.

How can researchers verify the specificity of signals detected with SPAC2H10.01 antibody?

Verifying signal specificity is crucial for reliable interpretation of results obtained with SPAC2H10.01 antibody. Several complementary approaches can be implemented to confirm specificity. First, genetic validation using SPAC2H10.01 knockout or knockdown strains provides the most definitive control—specific signals should be absent or significantly reduced in these samples. Second, perform epitope competition assays by pre-incubating the antibody with excess purified SPAC2H10.01 antigen (using the 200μg antigen provided as positive control ) before application to samples, which should significantly diminish specific signals.

Additional verification can come from detecting the protein at the expected molecular weight by Western blot, though post-translational modifications may alter apparent size. Using multiple antibodies targeting different epitopes of the same protein (if available) can provide convergent evidence of specificity. For critical applications, mass spectrometry analysis of immunoprecipitated proteins can definitively identify the detected protein and any cross-reactive species.

When possible, complement antibody-based detection with orthogonal approaches such as RT-PCR or RNA-seq to correlate protein detection with mRNA expression levels, or fluorescent protein tagging to confirm localization patterns observed with immunofluorescence. These multiple lines of evidence collectively strengthen confidence in the specificity of signals obtained with SPAC2H10.01 antibody.

How should researchers interpret variations in SPAC2H10.01 detection across different experimental conditions?

Variations in SPAC2H10.01 detection across experimental conditions should be interpreted within the context of appropriate controls and technical considerations. First, ensure that loading controls (such as actin or tubulin for Western blots) demonstrate equal sample loading across conditions. Quantification of signals should use digital image analysis with appropriate background subtraction, and multiple biological replicates should be performed to establish statistical significance of observed variations.

Changes in SPAC2H10.01 detection may reflect actual biological regulation of the protein (altered expression, degradation, or localization) or technical variability. To distinguish between these possibilities, researchers should establish the reproducibility of the observation across independent experiments and different detection methods. Consider whether post-translational modifications might affect antibody recognition—changes in protein modification state rather than absolute abundance could explain some variation in detection.

When comparing different strains or treatments, consider whether the experimental manipulation itself might alter the accessibility of the protein to extraction procedures. For instance, changes in cell wall composition or integrity could affect protein extraction efficiency. In such cases, alternative extraction methods should be tested to ensure comparable protein recovery across conditions.

How can SPAC2H10.01 antibody be used in co-immunoprecipitation studies to identify protein interaction partners?

Co-immunoprecipitation (Co-IP) experiments with SPAC2H10.01 antibody can reveal physiologically relevant protein interactions, providing insights into the protein's function within cellular networks. For successful Co-IP, gentle lysis conditions that preserve protein-protein interactions are essential. Non-denaturing detergents like NP-40 or Digitonin at 0.5-1% in buffers with physiological salt concentrations (120-150mM NaCl) are typically effective for yeast samples.

The SPAC2H10.01 antibody can be coupled to a solid support such as Protein A/G beads for direct immunoprecipitation. Alternatively, the antibody can be used in conjunction with magnetic beads conjugated with anti-rabbit IgG. Pre-clearing lysates with beads alone before adding the specific antibody reduces non-specific binding. After immunoprecipitation, interacting proteins can be identified by mass spectrometry analysis, which provides an unbiased approach to discovering novel interaction partners.

When designing Co-IP experiments, several controls are critical: (1) a negative control using pre-immune serum to identify non-specific interactions, (2) a reciprocal Co-IP with antibodies against suspected interaction partners to confirm the interaction bidirectionally, and (3) nuclease treatment of lysates to exclude DNA/RNA-mediated interactions. For detecting known or suspected interaction partners, Western blotting of Co-IP samples with specific antibodies against those proteins provides targeted confirmation of interactions.

What approaches can be used to study SPAC2H10.01 localization in fission yeast cells?

Studying SPAC2H10.01 localization provides critical insights into its function within cellular compartments and processes. While immunofluorescence (IF) microscopy using the SPAC2H10.01 antibody is a potential approach, this application would require validation as it is not explicitly listed among the validated applications . For IF, fixation conditions need careful optimization—formaldehyde fixation (3-4%) for 30-60 minutes is often suitable for yeast cells. Cell wall digestion is crucial for antibody penetration, using enzymatic cocktails containing zymolyase or lysing enzymes specific for S. pombe cell walls.

Complementary approaches include expressing SPAC2H10.01 with fluorescent protein tags (GFP, mCherry) for live-cell imaging. This approach avoids potential fixation artifacts and allows dynamic tracking of the protein. When tagging the protein, careful consideration of tag position (N- or C-terminal) is necessary to avoid interfering with localization signals or protein function.

Biochemical fractionation followed by Western blotting with the SPAC2H10.01 antibody provides an orthogonal method to verify subcellular localization. This involves separating cellular components (cytosol, membrane, nuclear, and cell wall fractions) through differential centrifugation and detergent treatments, followed by probing each fraction for the presence of SPAC2H10.01. Markers for different cellular compartments (e.g., histone H3 for nucleus, Pma1 for plasma membrane) should be included as controls for fractionation quality.

How can SPAC2H10.01 antibody be utilized in studies of protein post-translational modifications?

Investigating post-translational modifications (PTMs) of SPAC2H10.01 can reveal regulatory mechanisms controlling its function. The SPAC2H10.01 antibody can be used in immunoprecipitation to enrich the protein for subsequent analysis of PTMs by techniques such as mass spectrometry or Western blotting with modification-specific antibodies. When designing such experiments, phosphatase and deacetylase inhibitors should be included in lysis buffers to preserve labile modifications.

For glycosylation studies, which are particularly relevant for cell wall-associated proteins in yeast, treatment of immunoprecipitated SPAC2H10.01 with specific glycosidases (such as EndoH for N-linked glycans ) followed by Western blotting can reveal the presence and extent of glycosylation through mobility shifts. PAS-Silver staining methods can also be employed to specifically detect glycoproteins in gel-separated samples .

To study dynamics of PTMs, combining the SPAC2H10.01 antibody with phospho-specific or other modification-specific antibodies in sequential immunoblotting can track changes in modification status under different conditions. Alternatively, two-dimensional gel electrophoresis (separating by isoelectric point and molecular weight) followed by Western blotting can resolve differently modified forms of the protein. For comprehensive PTM mapping, mass spectrometry analysis of immunoprecipitated SPAC2H10.01 provides the most detailed information, identifying specific residues modified and potentially quantifying modification stoichiometry.