SPAC4C5.01 Antibody

What is SPAC4C5.01 and what organism does it originate from?

SPAC4C5.01 is a protein encoded by the SPAC4C5.01 gene in Schizosaccharomyces pombe (strain 972 / ATCC 24843), commonly known as fission yeast. This protein is identified by the UniProt accession number O14165. Antibodies against this protein are used in various research applications to study protein expression, localization, and function in this model organism. Fission yeast serves as an excellent eukaryotic model system for studying fundamental cellular processes due to its genetic tractability and similarities to human cells in terms of cell cycle regulation, chromosome dynamics, and other cellular mechanisms .

What validation methods should be used to confirm SPAC4C5.01 antibody specificity?

Validating antibody specificity for SPAC4C5.01 requires multiple complementary approaches. Begin with Western blotting against wild-type S. pombe lysates, comparing against a SPAC4C5.01 deletion strain (if available) as a negative control. Follow with immunoprecipitation coupled with mass spectrometry to confirm the antibody pulls down the target protein. Immunofluorescence microscopy comparing wild-type and knockout strains provides spatial validation. For definitive validation, perform epitope mapping through overlapping peptide arrays to identify the precise binding region. These methods collectively ensure the antibody specifically recognizes SPAC4C5.01 without cross-reactivity, mirroring approaches used in validating other research antibodies .

What are the recommended sample preparation protocols for using SPAC4C5.01 antibodies in immunoblotting?

For optimal results with SPAC4C5.01 antibodies in immunoblotting, harvest S. pombe cells during logarithmic growth phase and prepare lysates using mechanical disruption (glass beads) in a lysis buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and protease inhibitors. Include phosphatase inhibitors if phosphorylation states are relevant. After cell disruption, centrifuge at 13,000 × g for 15 minutes at 4°C to clear the lysate. For SDS-PAGE, load 20-50 μg of total protein per lane and transfer to a PVDF membrane. Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature. Incubate with SPAC4C5.01 antibody (CSB-PA522647XA01SXV) at a 1:1000 dilution overnight at 4°C. This protocol ensures consistent and specific detection of the target protein while minimizing background signal .

What positive and negative controls should be included when using SPAC4C5.01 antibodies?

When working with SPAC4C5.01 antibodies, implement a comprehensive control strategy to ensure experimental validity. For positive controls, use wild-type S. pombe (strain 972) lysates or purified recombinant SPAC4C5.01 protein if available. For negative controls, include: (1) SPAC4C5.01 deletion strain lysates, (2) pre-immune serum control at the same concentration as the primary antibody, (3) secondary antibody-only control to assess non-specific binding, and (4) competing peptide controls where the antibody is pre-incubated with excess antigenic peptide before application. When performing immunofluorescence, include an isotype control antibody matched to the SPAC4C5.01 antibody. This multi-layered approach to controls, similar to strategies employed in antibody validation studies for other targets, ensures that observed signals are specifically attributed to SPAC4C5.01 protein .

How should researchers optimize immunofluorescence protocols for SPAC4C5.01 antibody in fission yeast?

For optimal immunofluorescence with SPAC4C5.01 antibody in fission yeast, cells should be fixed with 3.7% formaldehyde for 30 minutes, followed by cell wall digestion with 0.5 mg/ml Zymolyase 100T for 30-60 minutes at 37°C. After permeabilization with 1% Triton X-100 for 5 minutes, block with 5% BSA in PBS for 1 hour. Apply primary SPAC4C5.01 antibody (CSB-PA522647XA01SXV) at 1:100-1:500 dilution overnight at 4°C, followed by fluorophore-conjugated secondary antibody at 1:500 for 1 hour at room temperature. Critical optimization parameters include fixation time (adjust between 15-45 minutes), enzymatic digestion duration (titrate between 15-90 minutes based on strain), and antibody concentration (test dilutions between 1:50-1:500). Include appropriate counter-stains such as DAPI for nuclear visualization. This protocol ensures specific detection while preserving cellular architecture, allowing accurate localization studies of the SPAC4C5.01 protein .

What cross-reactivity concerns exist when using SPAC4C5.01 antibodies across different yeast species?

Cross-reactivity is a significant consideration when applying SPAC4C5.01 antibodies across different yeast species. The antibody (CSB-PA522647XA01SXV) is specifically raised against the S. pombe SPAC4C5.01 protein (UniProt: O14165). Based on sequence homology analysis, researchers should anticipate potential cross-reactivity with closely related proteins in other Schizosaccharomyces species but limited reactivity with distant relatives like Saccharomyces cerevisiae. To assess cross-reactivity empirically, perform Western blot analysis using lysates from multiple yeast species alongside the target S. pombe strain. If cross-species applications are planned, consider epitope sequence conservation analysis using bioinformatics tools to predict potential cross-reactivity. When reporting results, explicitly document the specificity testing performed and acknowledge any limitations in antibody specificity across species boundaries .

What are the recommended antibody dilutions for different experimental applications?

The recommended antibody dilutions for SPAC4C5.01 antibody (CSB-PA522647XA01SXV) vary by application. The following dilution ranges have been optimized based on experimental validation:

These recommendations serve as starting points; optimization for specific experimental conditions is advised. For novel applications, perform a dilution series to determine optimal antibody concentration, balancing specific signal strength against background. Always include appropriate controls at each dilution to validate specificity .

How can SPAC4C5.01 antibodies be used to study protein-protein interactions in fission yeast?

SPAC4C5.01 antibodies provide powerful tools for investigating protein-protein interactions through multiple complementary approaches. For co-immunoprecipitation studies, lyse S. pombe cells under non-denaturing conditions (50 mM HEPES pH 7.5, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA with protease inhibitors) and incubate cleared lysates with SPAC4C5.01 antibody (CSB-PA522647XA01SXV) immobilized on Protein A/G beads. After washing, analyze co-precipitated proteins by mass spectrometry or Western blotting with antibodies against suspected interaction partners.

For in situ interaction studies, employ proximity ligation assays (PLA) using the SPAC4C5.01 antibody paired with antibodies against candidate interacting proteins. This technique enables visualization of interactions within their subcellular context. Alternatively, implement FRET-based approaches using fluorophore-conjugated antibodies against SPAC4C5.01 and potential binding partners.

When reporting interaction data, rigorously control for non-specific binding through isotype controls, pre-immune serum controls, and validation in SPAC4C5.01 deletion strains. These methodologies parallel advanced protein interaction studies used with other target proteins in similar model systems .

What strategies can overcome epitope masking issues when detecting SPAC4C5.01 in different cellular compartments?

Epitope masking of SPAC4C5.01 in different cellular compartments presents a significant challenge that requires tailored approaches. When SPAC4C5.01 interactions or conformational changes obscure the epitope, implement multiple antigen retrieval strategies: (1) Heat-mediated retrieval in citrate buffer (pH 6.0) at 95°C for 10-20 minutes, (2) Enzymatic digestion with proteinase K (1-10 μg/ml for 5-15 minutes), or (3) Chemical retrieval using SDS treatment (0.5% SDS for 5 minutes) followed by extensive washing.

For challenging nuclear detection, use stronger permeabilization with 0.5% Triton X-100 for 15 minutes, combined with DNase I treatment (100 U/ml for 30 minutes) to reduce chromatin interference. When SPAC4C5.01 localizes to membrane-bound organelles, supplement standard protocols with gentle sonication (3 cycles of 10 seconds at 20% amplitude) to enhance antibody accessibility.

To comprehensively address epitope masking, employ multiple antibodies targeting different SPAC4C5.01 epitopes if available. This strategy, similar to approaches used in studies of complex protein localization, ensures detection regardless of which epitopes might be masked in specific cellular contexts .

How can researchers quantitatively analyze SPAC4C5.01 expression levels across different experimental conditions?

For rigorous quantitative analysis of SPAC4C5.01 expression across experimental conditions, researchers should implement a multi-method approach. Begin with quantitative Western blotting using the SPAC4C5.01 antibody (CSB-PA522647XA01SXV) alongside loading controls (α-tubulin or GAPDH). Capture images within the linear dynamic range using a digital imaging system and analyze band intensities with software like ImageJ or Bio-Rad Image Lab.

For higher throughput analysis, develop a sandwich ELISA using SPAC4C5.01 antibody as the capture antibody and a differently-epitoped SPAC4C5.01 antibody (if available) for detection. This allows precise quantification against a standard curve of recombinant SPAC4C5.01.

For single-cell resolution, perform quantitative immunofluorescence with careful attention to consistent imaging parameters (exposure time, gain, offset) across all samples. Apply automated image analysis to measure integrated fluorescence intensity normalized to cell area or volume.

For absolute quantification, implement Selected Reaction Monitoring (SRM) mass spectrometry using isotope-labeled peptide standards corresponding to SPAC4C5.01 regions. This provides the most accurate measure of protein abundance independent of antibody binding efficiency.

Regardless of method, statistical analysis should include multiple biological replicates (n≥3) and appropriate statistical tests (ANOVA with post-hoc comparisons for multiple conditions) to determine significance of observed differences .

What are the considerations for using SPAC4C5.01 antibodies in chromatin immunoprecipitation (ChIP) experiments?

When implementing ChIP protocols with SPAC4C5.01 antibodies, several critical factors must be addressed for successful outcomes. First, optimize crosslinking conditions specifically for S. pombe; start with 1% formaldehyde for 15 minutes at room temperature, but test a range (10-30 minutes) to maximize crosslinking efficiency without overfixation. For cell wall disruption, use both enzymatic (Zymolyase 100T at 1 mg/ml) and mechanical (glass bead beating) methods in combination.

For sonication, determine optimal conditions empirically to generate chromatin fragments between 200-500 bp; typically, 15-20 cycles of 30 seconds on/30 seconds off at 40% amplitude works well for S. pombe. Use 2-5 μg of SPAC4C5.01 antibody per ChIP reaction, but titrate to determine optimal amounts for your specific experimental system.

Include appropriate controls: input chromatin (pre-immunoprecipitation), mock IP with non-specific IgG, and ideally a SPAC4C5.01 deletion strain as a negative control. For ChIP-seq applications, ensure library preparation methods accommodate the typically lower yields from yeast ChIP samples. When analyzing data, account for the compact nature of the S. pombe genome, which may require modified peak-calling parameters compared to mammalian systems .

How should researchers address non-specific binding when using SPAC4C5.01 antibodies?

To address non-specific binding when using SPAC4C5.01 antibodies, implement a systematic optimization approach. First, increase blocking stringency by using 5% BSA or 5% non-fat milk in TBST with extended blocking times (2-3 hours at room temperature). If background persists, incorporate 0.1-0.5% Tween-20 in wash buffers and increase wash durations and frequencies (5-6 washes of 10 minutes each).

For Western blotting applications, dilute the SPAC4C5.01 antibody (CSB-PA522647XA01SXV) in fresh blocking buffer containing 0.05-0.1% SDS to reduce hydrophobic interactions. Pre-adsorption is another effective strategy: incubate the diluted antibody with lysates from SPAC4C5.01 deletion strains for 2 hours at 4°C before application to experimental samples.

For immunofluorescence applications, include 10% normal serum from the species in which the secondary antibody was raised, and add 0.1-0.3 M NaCl to antibody dilution buffers to increase ionic strength. If membranous or nuclear envelope staining shows high background, add 0.1% Triton X-100 to antibody dilution buffers.

Document all optimization steps and include representative images of positive and negative controls in publications to demonstrate specificity. These systematic approaches mirror strategies successfully employed to overcome non-specific binding with other research antibodies .

What are the common pitfalls in interpreting SPAC4C5.01 localization data from immunofluorescence studies?

When interpreting SPAC4C5.01 localization data from immunofluorescence studies, researchers must navigate several potential pitfalls. First, autofluorescence from yeast cell walls can be misinterpreted as peripheral protein localization; control for this by imaging unlabeled cells and SPAC4C5.01 deletion strains under identical conditions. The compact nature of S. pombe cells (typically 3-4 μm in diameter) makes it challenging to distinguish between different subcellular compartments without super-resolution microscopy or appropriate co-localization markers.

Fixation artifacts present another challenge, as different fixation methods can alter apparent protein localization. Compare methanol and formaldehyde fixation results, as methanol can extract membrane lipids and alter membrane protein localization. Cell cycle-dependent localization changes must be considered; synchronize cells or use cell cycle markers (such as septum formation or nuclear morphology) to stage individual cells within asynchronous populations.

How can researchers validate antibody-based findings about SPAC4C5.01 using orthogonal methods?

To rigorously validate antibody-based findings about SPAC4C5.01, researchers should implement multiple orthogonal approaches. For expression level findings, complement antibody-based detection with RT-qPCR to measure transcript abundance, fluorescent protein tagging (GFP/mCherry) of endogenous SPAC4C5.01, and targeted mass spectrometry for absolute protein quantification.

For localization claims, confirm antibody-based immunofluorescence results with live-cell imaging of fluorescently tagged SPAC4C5.01, subcellular fractionation followed by Western blotting, and proximity labeling approaches like BioID or APEX2. When validating protein-protein interactions, supplement co-immunoprecipitation findings with yeast two-hybrid assays, bimolecular fluorescence complementation (BiFC), or FRET/FLIM approaches.

For functional studies, genetic approaches offer powerful validation: compare phenotypes from SPAC4C5.01 deletion, point mutations, or conditional degradation systems. CRISPR/Cas9-mediated genome editing to introduce epitope tags or mutations can provide additional confirmation of antibody specificity and biological findings.

How can SPAC4C5.01 antibodies be adapted for super-resolution microscopy studies in yeast?

Adapting SPAC4C5.01 antibodies for super-resolution microscopy in yeast requires specific technical optimizations across multiple techniques. For STORM imaging, conjugate the SPAC4C5.01 antibody (CSB-PA522647XA01SXV) directly with photoswitchable fluorophores like Alexa Fluor 647 using commercial conjugation kits with a dye-to-antibody ratio of 1-2 to prevent fluorophore self-quenching. Alternatively, use secondary antibodies labeled with appropriate fluorophores.

For STED microscopy, select secondary antibodies conjugated with dyes optimized for depletion wavelengths of available STED systems (typically STAR 580, STAR RED). Sample preparation requires modified protocols: use thinner coverslips (No. 1.5H, 170 ± 5 μm) and mount samples in specialized media with matched refractive indices to minimize spherical aberrations.

Cell wall interference presents a unique challenge in yeast; optimize enzymatic digestion with Zymolyase (0.5-1.0 mg/ml for 30-60 minutes) followed by gentle permeabilization with reduced detergent concentrations (0.1% Triton X-100 for 5 minutes) to preserve ultrastructural details. For multi-color super-resolution, carefully select fluorophore combinations to minimize bleed-through and cross-talk.

When imaging, use S. pombe cells immobilized on ConA-coated coverslips and implement drift correction using fiducial markers (TetraSpeck beads). These optimizations enable nanoscale visualization of SPAC4C5.01 distribution, potentially revealing previously undetectable protein organization patterns .

What considerations are important when designing multiplexed assays that include SPAC4C5.01 antibodies?

When designing multiplexed assays incorporating SPAC4C5.01 antibodies, several critical factors must be addressed to ensure reliable results. First, antibody compatibility is essential; select SPAC4C5.01 antibody clones that differ in host species or isotype from other antibodies in the panel to enable selective secondary detection. If using directly conjugated primary antibodies, choose fluorophores with minimal spectral overlap and implement comprehensive spillover compensation controls.

For multiplexed immunofluorescence, sequential staining protocols often yield better results than simultaneous incubation; begin with the lowest abundance target (often SPAC4C5.01) and progress to more abundant proteins. Implement epitope retrieval between staining rounds if necessary, using mild glycine treatment (100 mM, pH 2.5 for 10 minutes) to strip previous antibodies while preserving tissue morphology.

For multiplex Western blotting, consider size separation of targets and use fluorescently labeled secondary antibodies with distinct emission spectra. Alternatively, implement sequential reprobing with stripping between antibodies, validating complete stripping by incubating with secondary antibody alone.

In flow cytometry applications, titrate each antibody individually before combining into panels, as optimal concentrations may shift in multiplex settings. Include fluorescence-minus-one (FMO) controls for each marker to set appropriate gates. For all multiplexed approaches, validate the assay thoroughly by comparing results with single-marker detection to ensure that multiplexing doesn't alter detection sensitivity or specificity .

How can researchers develop quantitative assays to measure SPAC4C5.01 protein modifications?

Developing quantitative assays for SPAC4C5.01 protein modifications requires sophisticated approaches targeting specific post-translational modifications (PTMs). For phosphorylation analysis, implement a two-pronged strategy: (1) Phospho-specific antibody development targeting predicted phosphorylation sites based on consensus motifs and phosphoproteomic data, and (2) Phos-tag SDS-PAGE followed by Western blotting with SPAC4C5.01 antibody (CSB-PA522647XA01SXV) to separate phosphorylated from non-phosphorylated forms.

For ubiquitination studies, perform immunoprecipitation with SPAC4C5.01 antibody followed by Western blotting with anti-ubiquitin antibodies, or conversely, pull down ubiquitinated proteins using TUBE (Tandem Ubiquitin Binding Entities) and probe for SPAC4C5.01. Implement AQUA (Absolute Quantification) peptide standards corresponding to modified and unmodified SPAC4C5.01 peptides for precise quantification by parallel reaction monitoring mass spectrometry.

For acetylation or methylation analysis, use pan-acetyl-lysine or pan-methyl-lysine antibodies after SPAC4C5.01 immunoprecipitation. Site-specific modification analysis requires targeted mass spectrometry approaches like Selected Reaction Monitoring (SRM) with isotope-labeled peptide standards incorporating the modification of interest.

When validating these assays, include appropriate controls: treatment with phosphatases, deubiquitinases, or deacetylases to demonstrate specificity; mutagenesis of predicted modification sites; and correlation with physiological states known to affect the modification. These approaches enable precise quantification of SPAC4C5.01 modifications under different experimental conditions .