SPBC1773.15 Antibody

What is SPBC1773.15 Antibody and what is its target protein?

SPBC1773.15 Antibody (CSB-PA530930XA01SXV) is a polyclonal antibody raised in rabbits against a recombinant Schizosaccharomyces pombe (strain 972/ATCC 24843) SPBC1773.15 protein . This antibody specifically targets the SPBC1773.15 protein, which is expressed in fission yeast (S. pombe). The antibody is produced through immunization of rabbits with purified recombinant protein, followed by antigen affinity purification to ensure specificity. SPBC1773.15 Antibody is manufactured in liquid form and is suitable for research applications only, not for diagnostic or therapeutic procedures. The antibody preparation contains 0.03% Proclin 300 as a preservative and is formulated in a buffer of 50% Glycerol and 0.01M PBS at pH 7.4 to maintain stability during storage and use .

What are the recommended applications for SPBC1773.15 Antibody?

SPBC1773.15 Antibody has been specifically tested and validated for enzyme-linked immunosorbent assay (ELISA) and Western blotting (WB) applications . For Western blotting, the antibody allows researchers to detect and analyze SPBC1773.15 protein expression in cell lysates or tissue samples after separation by SDS-PAGE and transfer to a membrane. In ELISA applications, the antibody can be used to quantitatively measure SPBC1773.15 protein levels in various sample types. These applications are particularly useful for studying protein expression patterns, post-translational modifications, and protein-protein interactions in fission yeast models. While not explicitly stated in the product information, experienced researchers might also adapt protocols for other potential applications such as immunoprecipitation, immunofluorescence, or flow cytometry after appropriate validation. When developing new applications, researchers should include proper controls to ensure specific antigen identification .

How should SPBC1773.15 Antibody be stored for optimal stability?

SPBC1773.15 Antibody should be stored at -20°C or -80°C immediately upon receipt to maintain its specificity and reactivity . Repeated freeze-thaw cycles should be strictly avoided as they can lead to protein denaturation, aggregation, and loss of antibody activity. The antibody is supplied in a solution containing 50% glycerol, which helps prevent complete freezing and minimizes damage during freeze-thaw cycles if they must occur. For working stocks that will be used frequently, small aliquots can be prepared and stored separately to avoid repeated freezing and thawing of the entire stock. When handling the antibody during experiments, it should be kept on ice or at 4°C and returned to -20°C or -80°C promptly after use. The preservative (0.03% Proclin 300) in the storage buffer helps prevent microbial contamination, but proper aseptic technique should still be employed when handling the antibody to prevent introducing contaminants .

What is the difference between monoclonal and polyclonal antibodies like SPBC1773.15?

SPBC1773.15 Antibody is classified as a polyclonal antibody, which means it contains a heterogeneous mixture of antibodies that recognize multiple epitopes on the SPBC1773.15 protein . Polyclonal antibodies are produced by immunizing animals (in this case, rabbits) with a specific antigen, which stimulates multiple B cell clones to produce different antibodies targeting various epitopes on the same antigen. This characteristic provides polyclonal antibodies with higher sensitivity compared to monoclonal antibodies, as they can bind to multiple sites on the target protein simultaneously. In contrast, monoclonal antibodies are derived from a single B cell clone and recognize only one specific epitope on the antigen. While monoclonal antibodies offer higher specificity for a particular epitope, they may be more susceptible to epitope masking or destruction during sample processing. For research applications involving SPBC1773.15, the polyclonal nature of this antibody provides advantages in detection sensitivity, particularly in Western blotting and ELISA applications where signal amplification is desirable .

How can I determine the appropriate dilution for SPBC1773.15 Antibody in my experiments?

Determining the optimal dilution for SPBC1773.15 Antibody requires systematic titration experiments specific to each application and experimental system. For Western blotting applications, researchers should begin with a dilution range of 1:500 to 1:2000 and perform a gradient dilution experiment to identify the concentration that provides the best signal-to-noise ratio. For ELISA applications, initial testing with dilutions ranging from 1:1000 to 1:5000 is recommended, followed by optimization based on signal intensity and background levels. When determining optimal dilutions, researchers should include appropriate positive and negative controls to accurately assess specificity and sensitivity. The optimization process should also consider the abundance of the target protein in samples, the detection system used (chemiluminescence, fluorescence, or colorimetric), and the blocking agent employed to reduce non-specific binding. For novel applications not explicitly listed in the product information, more extensive titration experiments starting with higher antibody concentrations (1:100 to 1:500) may be necessary to establish working parameters .

How can SPBC1773.15 Antibody be validated for specificity in Schizosaccharomyces pombe studies?

Validating SPBC1773.15 Antibody specificity is crucial for ensuring reliable research outcomes when studying Schizosaccharomyces pombe. A comprehensive validation approach should include multiple complementary methods. First, researchers should perform Western blot analysis using wild-type S. pombe lysates alongside a knockout or knockdown strain lacking SPBC1773.15 expression; the absence of the target band in the knockout sample provides strong evidence of antibody specificity. Second, pre-absorption tests can be conducted by incubating the antibody with excess purified recombinant SPBC1773.15 protein prior to immunoassays; diminished or absent signal after pre-absorption indicates specific binding. Third, peptide competition assays using synthetic peptides representing different regions of SPBC1773.15 can help identify the specific epitopes recognized by the antibody. Fourth, immunoprecipitation followed by mass spectrometry analysis can confirm whether the antibody pulls down the correct target protein and identify any cross-reactive proteins. Finally, correlation of protein detection with mRNA expression levels across different experimental conditions can provide additional validation of antibody specificity and performance .

What troubleshooting approaches are effective when SPBC1773.15 Antibody shows non-specific binding in Western blots?

When encountering non-specific binding with SPBC1773.15 Antibody in Western blots, several methodological adjustments can significantly improve results. First, optimize the blocking procedure by testing different blocking agents (BSA, non-fat dry milk, commercial blocking buffers) at various concentrations (3-5%) and increasing blocking time (1-3 hours at room temperature or overnight at 4°C). Second, modify antibody incubation conditions by reducing primary antibody concentration, adding 0.1-0.3% Tween-20 to dilution buffers, or performing incubations at 4°C overnight instead of at room temperature. Third, increase washing stringency by extending wash times, adding higher concentrations of detergent (0.1-0.5% Tween-20), or using PBS-T with 500mM NaCl for more stringent washes after antibody incubations. Fourth, sample preparation can be improved by using fresher samples, adding protease inhibitors during extraction, and ensuring complete protein denaturation before loading. Fifth, consider using more specific detection methods such as highly cross-adsorbed secondary antibodies or detection systems with lower background. Finally, gradient SDS-PAGE gels can improve protein separation and reduce non-specific bands, while optimization of transfer conditions (time, voltage, buffer composition) can enhance the specificity of protein transfer to membranes .

How can SPBC1773.15 Antibody be optimized for immunoprecipitation experiments in fission yeast studies?

Optimizing SPBC1773.15 Antibody for immunoprecipitation (IP) in fission yeast studies requires careful consideration of multiple experimental parameters. First, lysate preparation is critical – cells should be disrupted using methods that preserve protein-protein interactions, such as gentle mechanical disruption with glass beads in non-denaturing buffers containing appropriate protease inhibitors and low concentrations of mild detergents (0.1-0.5% NP-40 or Triton X-100). Second, pre-clearing the lysate with protein A/G beads for 1 hour at 4°C before adding the antibody can significantly reduce non-specific binding. Third, antibody concentration must be optimized; typically starting with 2-5 μg of antibody per 500 μg of total protein and adjusting based on results. Fourth, the antibody-antigen binding step should be performed overnight at 4°C with gentle rotation to maximize specific interactions while minimizing non-specific binding. Fifth, wash conditions require optimization, with increasingly stringent washes (from low to high salt concentrations) to remove non-specifically bound proteins while retaining specific interactions. Sixth, elution conditions should be optimized based on downstream applications; gentle elution with peptide competition or more stringent elution with SDS-containing buffers may be appropriate depending on experimental goals. Finally, appropriate controls including IgG isotype controls, pre-immune serum controls, and immunoprecipitation from strains lacking the target protein are essential for validating results .

What considerations should be made when using SPBC1773.15 Antibody in co-localization studies with other cellular markers?

When designing co-localization studies using SPBC1773.15 Antibody with other cellular markers in fission yeast, several critical factors must be addressed to ensure reliable results. First, fixation and permeabilization methods must be carefully selected to preserve cellular architecture while allowing antibody access to target epitopes; paraformaldehyde fixation (3-4%) followed by detergent permeabilization (0.1-0.5% Triton X-100) is often effective for S. pombe. Second, blocking procedures should be optimized to minimize background signal; 1-5% BSA or normal serum from the species of the secondary antibody for 30-60 minutes is typically effective. Third, when using multiple primary antibodies, they must be derived from different host species (e.g., rabbit anti-SPBC1773.15 combined with mouse anti-cellular marker) to allow for species-specific secondary antibody detection. Fourth, fluorophore selection requires careful consideration of spectral overlap, with widely separated excitation/emission spectra preferred to minimize bleed-through; appropriate single-label controls are essential for setting imaging parameters. Fifth, imaging parameters must be optimized for each fluorophore independently, including exposure time, gain, and offset settings. Sixth, quantitative co-localization analysis should employ multiple mathematical approaches (Pearson's correlation coefficient, Manders' overlap coefficient) and include appropriate controls such as biologically relevant positive controls and spatially distinct negative controls. Finally, 3D reconstruction may be necessary for accurate assessment of co-localization in the complex three-dimensional architecture of yeast cells .

What is the optimal Western blotting protocol for SPBC1773.15 Antibody?

The optimal Western blotting protocol for SPBC1773.15 Antibody involves several critical steps that must be carefully executed. Sample preparation should begin with efficient cell lysis in a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, and a complete protease inhibitor cocktail; approximately 20 μg of total protein per lane is typically sufficient for detection of SPBC1773.15. SDS-PAGE separation should use 10-12% acrylamide gels to provide optimal resolution of the target protein. Following electrophoresis, proteins should be transferred to nitrocellulose or PVDF membranes at 100V for 60-90 minutes in Towbin buffer (25 mM Tris, 192 mM glycine, 20% methanol). Membranes should be blocked with 5% non-fat dry milk in TBS-T (TBS with 0.1% Tween-20) for 1 hour at room temperature. The primary antibody (SPBC1773.15 Antibody) should be diluted in blocking buffer at 1:500 to 1:1000 and incubated overnight at 4°C with gentle agitation. After primary antibody incubation, membranes should be washed four times with TBS-T for 5 minutes each. HRP-conjugated anti-rabbit secondary antibody should be diluted 1:5000 in blocking buffer and incubated for 1 hour at room temperature. Following four additional 5-minute washes with TBS-T, chemiluminescent detection can be performed using standard ECL reagents with exposure times optimized based on signal intensity .

How should cells be fixed and permeabilized for optimal detection with SPBC1773.15 Antibody in immunofluorescence?

For optimal immunofluorescence detection using SPBC1773.15 Antibody in S. pombe cells, a carefully optimized fixation and permeabilization protocol is essential. Cells should first be grown to mid-log phase (OD600 = 0.5-0.8) and harvested by centrifugation at 1000 × g for 5 minutes. Fixation should be performed with freshly prepared 3.7% formaldehyde for 30 minutes at room temperature, followed by three washes with PEM buffer (100 mM PIPES pH 6.9, 1 mM EGTA, 1 mM MgSO4). Cell wall digestion is a critical step for antibody accessibility; treat cells with zymolyase (1 mg/ml in PEM buffer with 1.2 M sorbitol) for 20-30 minutes at 37°C, monitoring periodically for sufficient digestion (80-90% of cells becoming phase-dark under phase contrast microscopy). After cell wall digestion, cells should be gently pelleted and permeabilized with 1% Triton X-100 in PEM buffer for 5 minutes at room temperature. Following permeabilization, blocking should be performed with 5% BSA in PEMBAL buffer (PEM buffer with 1% BSA, 0.1% sodium azide, 100 mM lysine hydrochloride) for 30 minutes at room temperature. SPBC1773.15 Antibody should be diluted 1:100 to 1:250 in PEMBAL and incubated with cells overnight at 4°C. After primary antibody incubation, cells should be washed three times with PEMBAL before incubation with fluorescently labeled anti-rabbit secondary antibody (diluted 1:500) for 2 hours at room temperature in the dark. Finally, cells should be washed three times with PEMBAL, mounted in antifade mounting medium containing DAPI (1 μg/ml), and sealed with nail polish for imaging .

What protein extraction methods are most compatible with SPBC1773.15 Antibody detection?

Several protein extraction methods are highly compatible with SPBC1773.15 Antibody detection, each with specific advantages depending on the experimental context. The TCA precipitation method offers excellent protein recovery and preservation of post-translational modifications; cells should be harvested, washed with cold water, and resuspended in 20% TCA, followed by mechanical disruption with glass beads, neutralization with Tris base, and resuspension in SDS sample buffer. The alkaline extraction method is rapid and efficient; cells are treated with 0.3M NaOH for 10 minutes on ice, followed by TCA precipitation, acetone washing, and resuspension in SDS sample buffer with 0.1M Tris base. For native protein applications, a gentle extraction method using non-ionic detergents is preferred; cells should be disrupted in buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10% glycerol, and protease inhibitor cocktail. When studying membrane-associated proteins, inclusion of 0.5-1% sodium deoxycholate or 0.1% SDS in the extraction buffer can improve solubilization. For investigating nuclear proteins, extraction should include a nuclear isolation step using 0.25% Triton X-100 lysis followed by nuclear extraction with high-salt buffer (420 mM NaCl, 20 mM HEPES pH 7.9, 1.5 mM MgCl2, 0.2 mM EDTA, and 25% glycerol). All extraction methods should incorporate freshly prepared protease inhibitors (PMSF, leupeptin, aprotinin, pepstatin A) and phosphatase inhibitors if phosphorylation status is being investigated .

What are the recommended controls when using SPBC1773.15 Antibody in mechanistic studies of gene function?

Rigorous control strategies are essential when using SPBC1773.15 Antibody in mechanistic studies of gene function. Genetic controls should include wild-type strains alongside SPBC1773.15 deletion mutants (when viable) or conditional mutants (temperature-sensitive or repressible promoter constructs) to confirm antibody specificity. Expression controls using strains with tagged versions of SPBC1773.15 (e.g., GFP-tagged or epitope-tagged constructs) allow parallel detection with commercial anti-tag antibodies to validate expression patterns. Antibody controls should include primary antibody omission controls, isotype controls using non-specific rabbit IgG at the same concentration, and pre-immune serum controls when available. Competition controls where the primary antibody is pre-incubated with excess recombinant SPBC1773.15 protein before application can demonstrate binding specificity. Loading controls using antibodies against stable, constitutively expressed proteins (e.g., α-tubulin, PSTAIR) are essential for normalizing expression levels across samples. Cell cycle controls using synchronized cultures or known cell cycle phase markers help interpret SPBC1773.15 expression or modification patterns that may vary with cell cycle progression. Stress response controls examining SPBC1773.15 under various stress conditions (temperature, oxidative, osmotic) provide context for functional studies. Finally, interaction controls using negative controls in co-immunoprecipitation experiments (such as immunoprecipitation with non-specific IgG) help validate specific protein-protein interactions detected with SPBC1773.15 Antibody .