SPAP27G11.11c Antibody

What is SPAP27G11.11c and what are the key properties of antibodies targeting this protein?

SPAP27G11.11c is an uncharacterized protein found in the fission yeast Schizosaccharomyces pombe (strain 972 / ATCC 24843). The commercially available polyclonal antibody against this protein is derived from rabbit host immunized with recombinant SPAP27G11.11c protein and is primarily used for detecting the native protein in yeast samples . This antibody has been validated for applications including Western blot (WB) and ELISA, making it suitable for both qualitative and quantitative analyses . The antibody preparation typically includes purified antibodies via antigen affinity chromatography, pre-immune serum for negative control, and recombinant antigen that can serve as a positive control in experimental setups . According to supplier information, the antibody is stored at -20°C or -80°C for optimal stability and performance over time . The specificity of this antibody is limited to yeast species, with particular reactivity to S. pombe strains.

How should SPAP27G11.11c antibody be validated before use in experimental procedures?

Proper validation of SPAP27G11.11c antibody before experimental use should follow a systematic approach similar to that used for other research antibodies. Researchers should first perform Western blot analysis using both the recombinant antigen (provided with the antibody) and wild-type S. pombe lysate to confirm specific binding to the target protein of expected molecular weight . Inclusion of appropriate controls is essential - the pre-immune serum supplied with the antibody serves as an excellent negative control to identify any non-specific binding . For quantitative applications like ELISA, a standard curve should be established using serial dilutions of the recombinant antigen to determine the linear range of detection and sensitivity limits. Cross-reactivity testing against lysates from related yeast species or strains with SPAP27G11.11c knockouts (if available) provides additional validation of specificity. Following protocols similar to those established for well-characterized antibodies like those against CD11c (ITGAX), which include multiple validation methods across different applications, ensures reliable experimental outcomes .

What sample preparation techniques are recommended for optimal detection of SPAP27G11.11c in yeast samples?

Sample preparation for SPAP27G11.11c detection requires careful consideration of protein extraction methods appropriate for yeast cell wall disruption. For Western blot applications, mechanical disruption methods such as glass bead beating in the presence of protease inhibitors have proven effective for releasing intracellular proteins from S. pombe while preserving antigenic properties . The lysis buffer composition should be optimized to maintain protein stability and epitope integrity - typically containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% Triton X-100, and a protease inhibitor cocktail. When preparing samples for immunoprecipitation, gentler extraction methods may be preferred to maintain protein-protein interactions. Sample denaturation conditions for SDS-PAGE should be carefully optimized, as excessive heating or strong reducing conditions might destroy important epitopes. For immunofluorescence applications, fixation methods should be validated specifically for SPAP27G11.11c, as fixation artifacts can dramatically impact antibody recognition, similar to what has been observed with other antibodies in complex samples .

How can epitope mapping be performed to determine the specific binding regions of polyclonal SPAP27G11.11c antibodies?

Epitope mapping for polyclonal SPAP27G11.11c antibodies can be approached using techniques similar to those employed in studies of other antibodies. Peptide microarray analysis, as demonstrated in antigen-antibody studies, enables high-resolution mapping of binding regions by screening antibodies against overlapping peptide sequences covering the entire SPAP27G11.11c protein . This method would involve synthesizing overlapping peptides (typically 15-20 amino acids with 5-10 amino acid overlaps) spanning the full SPAP27G11.11c sequence, immobilizing them on a microarray surface, and probing with the polyclonal antibody to identify reactive peptides. Alternatively, a proteolytic fragmentation approach could be employed, wherein recombinant SPAP27G11.11c protein is subjected to limited proteolysis, the fragments separated by SDS-PAGE, transferred to membranes, and probed with the antibody to identify immunoreactive fragments . The identified fragments can then be analyzed by mass spectrometry to determine their sequence. For more precise mapping, alanine scanning mutagenesis of recombinant SPAP27G11.11c could be performed, introducing systematic alanine substitutions throughout the protein sequence to identify amino acid residues critical for antibody binding .

What strategies can be employed to improve signal-to-noise ratio when using SPAP27G11.11c antibody in immunofluorescence applications?

Improving signal-to-noise ratio with SPAP27G11.11c antibody in immunofluorescence requires specialized approaches for yeast cell imaging. First, optimization of fixation and permeabilization protocols is critical - testing various fixatives (formaldehyde, methanol, or glyoxal) and permeabilization agents (Triton X-100, digitonin, or enzymatic cell wall digestion with zymolyase) can significantly impact antibody accessibility to intracellular targets . Implementing a stringent blocking protocol using a combination of BSA (3-5%) and normal serum from the secondary antibody host species helps minimize non-specific binding. Signal amplification systems, such as tyramide signal amplification or quantum dot-conjugated secondary antibodies, can enhance detection of low-abundance proteins while maintaining favorable signal-to-noise ratios. Advanced microscopy techniques like structured illumination microscopy (SIM) or stimulated emission depletion (STED) microscopy can further improve spatial resolution and signal discrimination. Additionally, employing image acquisition parameters that optimize dynamic range while avoiding pixel saturation, followed by appropriate deconvolution algorithms, can significantly enhance signal quality in post-processing, similar to techniques used with other challenging cellular targets .

How can SPAP27G11.11c antibody be effectively employed in chromatin immunoprecipitation (ChIP) studies to investigate potential DNA-binding properties?

Adapting SPAP27G11.11c antibody for chromatin immunoprecipitation requires careful protocol optimization similar to established ChIP methods. Begin with crosslinking optimization by testing various formaldehyde concentrations (0.5-3%) and incubation times (5-20 minutes) to preserve protein-DNA interactions while maintaining epitope accessibility . Cell wall digestion with zymolyase or lyticase followed by gentle mechanical disruption is critical for efficient chromatin extraction from S. pombe without damaging the crosslinked complexes. Sonication conditions should be carefully calibrated to generate DNA fragments of 200-500 bp, verifying fragment size by agarose gel electrophoresis before proceeding. The immunoprecipitation step requires optimization of antibody concentration, typically starting with 2-5 μg per reaction, and incubation conditions (4°C overnight with rotation) . Including appropriate controls is essential: input chromatin (pre-immunoprecipitation sample), no-antibody control, and ideally a ChIP using pre-immune serum. For qPCR analysis of immunoprecipitated DNA, design primers targeting potential binding regions based on bioinformatic predictions or regions of interest in the S. pombe genome. Sequential ChIP (re-ChIP) may be employed to investigate co-occupancy of SPAP27G11.11c with other proteins at specific genomic loci .

How should dilution optimization be performed for SPAP27G11.11c antibody across different applications?

Systematic dilution optimization for SPAP27G11.11c antibody should follow a strategic titration approach across applications. For Western blot applications, begin with a concentration gradient ranging from 1:500 to 1:5000 dilutions of the antibody, using both the recombinant antigen and native S. pombe lysates as test samples . Each dilution should be evaluated based on signal intensity, background levels, and the appearance of non-specific bands. For ELISA applications, prepare a matrix of coating antigen concentrations versus antibody dilutions to determine optimal combinations, typically starting with coating concentrations of 0.1-10 μg/ml and antibody dilutions from 1:1000 to 1:20,000 . For each application, signal-to-noise ratio calculations provide quantitative assessment of optimal conditions, with values above 5:1 generally considered acceptable for research applications. Temperature and incubation time variations (1-hour room temperature versus overnight at 4°C) should be systematically tested to determine conditions that maximize specific binding while minimizing background. All optimization experiments should include the pre-immune serum control at matching dilutions to properly account for non-specific binding .

What are the recommended protocols for using SPAP27G11.11c antibody in co-immunoprecipitation studies to identify protein interaction partners?

Co-immunoprecipitation with SPAP27G11.11c antibody requires gentle lysis conditions to preserve native protein complexes. A recommended lysis buffer composition includes 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% NP-40 or 1% digitonin, 5 mM EDTA, 5 mM EGTA, supplemented with protease and phosphatase inhibitors . For S. pombe cells, gentle mechanical disruption methods such as French press or glass bead vortexing with cooling intervals prevent protein complex disruption during extraction. Pre-clearing the lysate with Protein A/G beads for 1 hour at 4°C reduces non-specific binding. For immunoprecipitation, 2-5 μg of SPAP27G11.11c antibody should be incubated with pre-cleared lysate overnight at 4°C with gentle rotation, followed by addition of Protein A/G beads for 2-4 hours . Stringent washing steps with decreasing salt concentrations help preserve specific interactions while removing contaminants. Elution can be performed using either low pH glycine buffer (pH 2.8) followed by immediate neutralization or by boiling in SDS sample buffer. Analysis of co-immunoprecipitated proteins by mass spectrometry provides unbiased identification of interaction partners, while targeted Western blot can confirm suspected interactions with known proteins .

What controls and validation methods are essential when developing quantitative assays using SPAP27G11.11c antibody?

Rigorous controls and validation methods are crucial for quantitative assays with SPAP27G11.11c antibody. Standard curves using purified recombinant SPAP27G11.11c protein at concentrations spanning the expected physiological range (typically 0.1-100 ng/ml) should be included in each assay run to establish linearity, sensitivity, and reproducibility parameters . The pre-immune serum provided with the antibody serves as an essential negative control for establishing background signal levels and non-specific binding thresholds. Specificity validation should include testing against S. pombe strains with SPAP27G11.11c gene deletion or knockdown (if available) to confirm signal absence. For absolute quantification, spike-and-recovery experiments with known quantities of recombinant protein added to biological samples help assess matrix effects and extraction efficiency. Inter-assay and intra-assay coefficient of variation (CV) should be determined through replicate measurements, with CV values below 15% generally considered acceptable for research applications . Cross-reactivity testing against related yeast proteins helps establish specificity boundaries and potential false-positive scenarios. Reference samples of known concentration should be included in each assay run as quality control checkpoints to monitor assay drift over time .

What strategies can address non-specific binding issues when using SPAP27G11.11c antibody in complex yeast lysates?

Non-specific binding with SPAP27G11.11c antibody can be addressed through multiple optimization strategies. Implementing more stringent blocking protocols with combinations of 5% BSA, 5% non-fat dry milk, and 1-2% normal serum from the secondary antibody host species can significantly reduce background in immunoblotting and immunofluorescence applications . Increasing the detergent concentration (0.1-0.3% Tween-20 or 0.1% Triton X-100) in washing buffers helps disrupt weak, non-specific interactions while preserving the higher-affinity specific antibody-antigen binding. Pre-adsorption of the antibody with acetone powder prepared from non-target yeast species can remove cross-reactive antibodies from the polyclonal mixture . For particularly challenging samples, implementing a sequential epitope-exposure protocol with multiple blocking steps between primary and secondary antibody incubations can provide incremental improvements in specificity. Titrating salt concentration in washing and binding buffers (from 150 mM to 500 mM NaCl) can help optimize the electrostatic component of antibody-antigen interactions. Using competitive blocking with excess recombinant SPAP27G11.11c protein can confirm binding specificity, as true signals should be competitively inhibited while non-specific signals often remain .

How can SPAP27G11.11c antibody be effectively conjugated to fluorophores or enzymes for direct detection applications?

Direct conjugation of SPAP27G11.11c antibody to detection molecules follows protocols similar to those used for other antibodies like CD11c conjugates . For fluorophore conjugation, commercially available labeling kits utilizing NHS-ester chemistry can be employed to attach fluorophores like Alexa Fluor 488, 555, or 647 to primary amines on the antibody. The antibody concentration should be adjusted to 1-2 mg/ml in carbonate buffer (pH 8.3-8.5) before conjugation to ensure optimal labeling efficiency. A molar ratio of 4-10 fluorophore molecules per antibody generally provides good signal without compromising binding activity . Purification of conjugated antibody from unreacted fluorophore is critical and can be achieved using size exclusion chromatography or provided purification columns in commercial kits. For enzyme conjugation, maleimide chemistry targeting reduced disulfide bonds offers site-specific labeling that minimizes impact on the antigen-binding region. Horseradish peroxidase (HRP) or alkaline phosphatase (AP) conjugation typically employs periodate oxidation followed by reductive amination. After conjugation, activity retention should be verified by comparing the conjugated antibody with the unconjugated version in parallel assays to ensure that labeling has not compromised antigen recognition .

What computational approaches can predict potential cross-reactivity of SPAP27G11.11c antibody with homologous proteins in related species?

Computational prediction of SPAP27G11.11c antibody cross-reactivity involves multi-faceted bioinformatic analyses. Sequence homology assessment should begin with BLAST searches of the SPAP27G11.11c protein sequence against proteome databases of related yeast species and model organisms, focusing on proteins with >40% sequence identity or >60% similarity as potential cross-reactivity candidates . Epitope prediction algorithms such as BepiPred, ABCpred, or Ellipro can identify potential linear and conformational epitopes on the SPAP27G11.11c protein, which can then be aligned with homologous regions in related proteins to evaluate conservation at epitope sites . Structural modeling using homology-based approaches (SWISS-MODEL, I-TASSER) or AlphaFold2 predictions can generate three-dimensional models of SPAP27G11.11c and its homologs, allowing spatial comparison of surface-exposed epitopes. Molecular docking simulations between predicted antibody structures and potential cross-reactive proteins provide insights into binding energetics and interface complementarity . Phylogenetic analysis across yeast species creates an evolutionary framework for predicting cross-reactivity patterns based on evolutionary distance. Post-translational modification prediction tools should be employed to identify species-specific modifications that might affect epitope recognition in different organisms .

How can SPAP27G11.11c antibody be incorporated into high-throughput screening or multiplex detection systems?

Integration of SPAP27G11.11c antibody into high-throughput and multiplex systems requires adaptation of standard protocols to automated platforms. For microarray applications, the antibody can be spotted onto nitrocellulose or glass slides at concentrations of 0.1-1 mg/ml using commercial microarray printing systems, allowing simultaneous detection of SPAP27G11.11c alongside other proteins of interest . In bead-based multiplex assays, the antibody can be conjugated to distinctly coded microspheres (such as Luminex xMAP technology) using standard carbodiimide chemistry, with approximately 5-10 μg antibody per million beads. For automated ELISA systems, the antibody concentration and incubation parameters should be optimized specifically for robotic liquid handling dynamics, which may differ from manual protocols . When developing multiplex immunofluorescence panels, careful selection of compatible fluorophores with minimal spectral overlap is essential, similar to approaches used in multi-color flow cytometry. Testing for antibody cross-reactivity and competitive binding when used in combination with other antibodies is crucial for multiplex applications. Barcode-antibody conjugates, as employed in technologies like DNA-barcoded antibody sensing, offer another avenue for including SPAP27G11.11c detection in highly multiplexed systems .

How can SPAP27G11.11c antibody be used to investigate protein localization changes during cell cycle progression in S. pombe?

Investigating SPAP27G11.11c localization during cell cycle progression requires synchronized cell populations and high-resolution imaging techniques. Cell synchronization can be achieved using hydroxyurea block and release, lactose gradient centrifugation, or temperature-sensitive cdc mutants depending on the specific cell cycle phases of interest . Immunofluorescence microscopy with the SPAP27G11.11c antibody should be performed at defined time points after synchronization (typically every 15-30 minutes covering a complete cell cycle of 2-3 hours for S. pombe). Co-staining with established cell cycle markers such as Sad1 (spindle pole body), tubulin (mitotic spindle), or DAPI (DNA condensation state) provides critical reference points for cell cycle staging . For dynamic studies, time-lapse imaging in fixed cells from synchronized populations can be complemented with precise cell morphology measurements as S. pombe maintains consistent relationships between cell length and cell cycle position. Quantitative image analysis should include measurement of signal intensity, subcellular distribution patterns, and co-localization coefficients with organelle markers across different cell cycle stages. Advanced techniques such as fluorescence recovery after photobleaching (FRAP) or fluorescence correlation spectroscopy (FCS) with fluorophore-conjugated SPAP27G11.11c antibody in permeabilized cells can provide insights into protein mobility changes during cell cycle progression .

What approaches can be used to determine the affinity and binding kinetics of SPAP27G11.11c antibody to its target epitope?

Affinity and binding kinetics determination for SPAP27G11.11c antibody requires biophysical characterization techniques. Surface Plasmon Resonance (SPR) provides a comprehensive kinetic profile by immobilizing either the antibody or recombinant SPAP27G11.11c protein on a sensor chip and flowing the binding partner at various concentrations to measure association (ka) and dissociation (kd) rate constants, with equilibrium dissociation constant (KD) calculation (KD = kd/ka) . Bio-Layer Interferometry (BLI) offers similar kinetic information with the advantage of not requiring microfluidic systems, making it suitable for crude samples or high-throughput screening. Isothermal Titration Calorimetry (ITC) provides thermodynamic parameters (ΔH, ΔS, ΔG) alongside affinity constants, offering insights into the energetic basis of the antibody-antigen interaction. For polyclonal preparations, heterogeneity analysis should be performed to determine if the antibody population contains multiple affinities, which can be accomplished through Scatchard plot analysis of ELISA data or through SPR using different regeneration conditions to distinguish antibody subpopulations . Competitive ELISA with defined epitope peptides can provide epitope-specific affinity information when complete kinetic characterization is not feasible .

How can SPAP27G11.11c antibody be utilized in proteomic studies to investigate post-translational modifications of the target protein?

SPAP27G11.11c antibody can be effectively employed in proteomic workflows to characterize post-translational modifications (PTMs) through several approaches. Immunoprecipitation using the antibody followed by mass spectrometry analysis allows for enrichment of the target protein and its modified forms from complex cellular extracts . Optimization of extraction buffers to preserve labile PTMs is critical, with phosphatase inhibitors (sodium orthovanadate, sodium fluoride, β-glycerophosphate) for phosphorylation studies, deacetylase inhibitors (trichostatin A, nicotinamide) for acetylation studies, or N-ethylmaleimide for capturing thiol modifications. Following immunoprecipitation, on-bead or in-gel digestion with multiple proteases (trypsin, chymotrypsin, Glu-C) increases sequence coverage for comprehensive PTM mapping . For specific PTM investigations, the immunoprecipitated protein can be probed with PTM-specific antibodies (anti-phospho, anti-acetyl, anti-ubiquitin) in Western blots. Alternative enrichment strategies can complement antibody-based approaches, such as metal oxide affinity chromatography (MOAC) for phosphopeptides or lectin affinity for glycosylated forms, applied to immunoprecipitated material. Targeted mass spectrometry approaches like parallel reaction monitoring (PRM) or multiple reaction monitoring (MRM) can be developed for quantitative analysis of specific modified peptides identified in discovery-phase experiments .

What lessons from monoclonal antibody development can be applied to improve SPAP27G11.11c antibody characterization?

Lessons from monoclonal antibody development offer valuable insights for enhancing SPAP27G11.11c antibody characterization. The systematic epitope mapping approaches used in therapeutic antibody development, such as those employed for 1N11 and 2AF11 , could be adapted to identify the precise binding regions of polyclonal SPAP27G11.11c antibodies, potentially leading to more specific second-generation reagents. Rigorous cross-reactivity testing methodologies, as demonstrated in studies with anti-Siglec-11 antibodies , should be applied to comprehensively define the specificity profile across related yeast proteins and other fungal species. The implementation of standardized reporting frameworks like the Antibody Registry identifiers or structured validation protocols similar to those used for therapeutic antibodies would improve reproducibility across research groups . Advanced affinity maturation techniques, such as those used to develop high-affinity monoclonal antibodies like 24D11 , could inform the development of improved SPAP27G11.11c antibodies with enhanced sensitivity and specificity. Thorough application-specific validation workflows, similar to those performed for clinical diagnostic antibodies, would provide researchers with clearer guidelines for optimal use conditions across different experimental contexts .

Table 1: Recommended Dilution Ranges for SPAP27G11.11c Antibody Applications

Table 2: Comparison of Detection Methods for SPAP27G11.11c Protein