SPAC13F5.07c Antibody

What is SPAC13F5.07c protein and why is it studied in fission yeast? (Basic)

SPAC13F5.07c (UniProt ID: O13706) is a protein found in Schizosaccharomyces pombe (fission yeast), which serves as an important model organism for studying eukaryotic molecular and cellular biology. This protein is of particular interest because S. pombe's cellular processes closely resemble those in higher eukaryotes, making it valuable for understanding conserved mechanisms. When investigating this protein, researchers typically use polyclonal antibodies raised against recombinant SPAC13F5.07c protein to detect its expression, localization, and interactions within cellular networks .

How is the specificity of SPAC13F5.07c polyclonal antibody validated? (Advanced)

Validation of SPAC13F5.07c antibody specificity involves multiple complementary approaches:

• Western blot analysis with positive and negative controls: Testing the antibody against wild-type S. pombe lysates versus SPAC13F5.07c knockout strains

• Peptide competition assays: Pre-incubating the antibody with excess immunizing peptide to confirm signal reduction

• Cross-reactivity testing: Evaluating potential cross-reactivity with closely related proteins

• Immunoprecipitation followed by mass spectrometry: Confirming the identity of captured proteins

Researchers should note that antigen affinity purification methods, as used for this antibody, enhance specificity by removing non-specific antibodies from the polyclonal mixture .

What are the differences between mouse and rabbit-derived SPAC13F5.07c antibodies for research applications? (Advanced)

While the SPAC13F5.07c antibody described in the search results is rabbit-derived, this comparison is important for research planning:

For SPAC13F5.07c studies, rabbit polyclonals are often preferred due to their robust response to yeast proteins and multiple epitope recognition .

What are the validated applications for SPAC13F5.07c antibody? (Basic)

The SPAC13F5.07c polyclonal antibody has been validated for several applications in S. pombe research:

• ELISA (Enzyme-Linked Immunosorbent Assay): For quantitative detection of SPAC13F5.07c protein

• Western Blot (WB): For detection of denatured SPAC13F5.07c protein from cell lysates

When considering experimental applications, researchers should note that while these are the validated applications, optimization may allow use in additional techniques such as immunoprecipitation or immunofluorescence, though this would require additional validation .

How should optimal antibody dilutions be determined for different applications? (Advanced)

Determining optimal antibody dilutions for SPAC13F5.07c research requires systematic titration:

Western Blot Optimization Protocol:

• Prepare a dilution series (e.g., 1:500, 1:1000, 1:2000, 1:5000)

• Run identical protein samples on multiple gel lanes

• Process all blots simultaneously with varying antibody concentrations

• Assess signal-to-noise ratio quantitatively (using imaging software)

• Select the dilution with highest specific signal and lowest background

ELISA Optimization Approach:

• Perform checkerboard titration with both coating antigen and antibody dilutions

• Calculate signal-to-noise ratios for each combination

• Plot sensitivity curves to identify the optimal working range

For SPAC13F5.07c antibody, initial testing should start at manufacturer-recommended dilutions, then adjust based on signal strength and background levels .

How can SPAC13F5.07c antibody be used to investigate protein-protein interactions in fission yeast? (Advanced)

When investigating protein-protein interactions involving SPAC13F5.07c, several methodological approaches are effective:

• Co-immunoprecipitation (Co-IP):

  • Lyse cells in non-denaturing buffer (e.g., 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40)

  • Pre-clear lysate with protein A/G beads

  • Incubate with SPAC13F5.07c antibody (typically 2-5 μg per mg of protein)

  • Capture complexes with fresh protein A/G beads

  • Analyze interacting partners by mass spectrometry or Western blotting

• Proximity Ligation Assay (PLA):

  • Fix S. pombe cells with 4% paraformaldehyde

  • Permeabilize with 0.1% Triton X-100

  • Incubate with SPAC13F5.07c antibody and antibody against potential interactor

  • Apply PLA probes and perform ligation and amplification

  • Visualize interaction signals by fluorescence microscopy

These techniques must be carefully controlled using non-specific IgG and samples lacking the protein of interest .

What are common challenges when using SPAC13F5.07c antibody in Western blotting? (Basic)

Researchers commonly encounter these challenges when using SPAC13F5.07c antibody for Western blotting:

• High background: Often caused by insufficient blocking or excessive antibody concentration

• Weak signal: May result from low protein expression, inefficient transfer, or suboptimal antibody dilution

• Multiple bands: Could indicate protein degradation, post-translational modifications, or non-specific binding

• No signal: Potentially due to protein denaturation affecting epitope recognition or improper secondary antibody selection

For the SPAC13F5.07c antibody specifically, using 5% non-fat dry milk in TBST for blocking and ensuring proper storage at -20°C or -80°C can mitigate many of these issues .

How can cross-reactivity issues with SPAC13F5.07c antibody be addressed? (Advanced)

Cross-reactivity challenges can be systematically addressed through these methodological approaches:

• Pre-adsorption protocol:

  • Incubate antibody with 5-10× excess of recombinant proteins with sequence similarity

  • Allow binding for 2 hours at room temperature

  • Use the pre-adsorbed antibody in your experiment

  • Compare results with non-adsorbed controls

• Sequential immunodepletion:

  • Immobilize potential cross-reactive proteins on a solid support

  • Pass SPAC13F5.07c antibody through the column to remove cross-reactive antibodies

  • Validate specificity of the depleted antibody preparation

• Knockout/knockdown validation:

  • Test antibody reactivity in SPAC13F5.07c deletion strains

  • Any remaining signal indicates cross-reactivity

When working with yeast systems, additional washing steps (4-5 washes of 10 minutes each) with 0.1% Tween-20 in PBS can significantly reduce non-specific binding .

What methods can improve detection sensitivity when working with low-abundance SPAC13F5.07c protein? (Advanced)

For detecting low-abundance SPAC13F5.07c protein, consider these methodological enhancements:

• Signal amplification strategies:

  • Use enhanced chemiluminescence (ECL) substrates with femtogram sensitivity

  • Implement tyramide signal amplification (TSA) for immunodetection

  • Consider fluorescent secondary antibodies with direct scanning detection

• Sample enrichment techniques:

  • Perform immunoprecipitation before Western blotting

  • Use subcellular fractionation to concentrate the compartment containing SPAC13F5.07c

  • Apply TCA precipitation to concentrate proteins from dilute samples

• Protocol modifications:

  • Extend primary antibody incubation to overnight at 4°C

  • Use protein A-HRP instead of conventional secondary antibodies

  • Incorporate 0.1% SDS in antibody dilution buffer to enhance accessibility

These approaches have demonstrated 5-10 fold improvement in detection sensitivity in challenging yeast protein detection scenarios .

What are the optimal storage conditions for SPAC13F5.07c antibody? (Basic)

The SPAC13F5.07c antibody requires specific storage conditions to maintain its activity:

• Store at -20°C or -80°C for long-term stability

• Avoid repeated freeze-thaw cycles as they can denature antibody proteins

• Aliquot the antibody solution into single-use volumes upon receipt

• The antibody is supplied in a storage buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative

• For working solutions, store at 4°C for up to one week

When handling the antibody, minimize exposure to room temperature and use sterile technique to prevent microbial contamination .

How does the buffer composition affect SPAC13F5.07c antibody performance? (Advanced)

The buffer composition significantly impacts SPAC13F5.07c antibody performance in multiple ways:

For certain applications, buffer exchange may be necessary:

• For enzyme-linked assays sensitive to preservatives, dialyze against preservative-free buffer

• For immunoprecipitation, dilute at least 1:10 in IP buffer to reduce glycerol concentration

• For mass spectrometry applications, consider antibody purification to remove buffer components that may interfere with analysis .

What quality control metrics should be monitored for antibody performance over time? (Advanced)

To monitor SPAC13F5.07c antibody performance over time, implement these quality control procedures:

• Reference sample testing:

  • Maintain aliquots of a characterized positive control sample

  • Test new and stored antibody lots against this reference monthly

  • Document band intensity, background levels, and specific/non-specific signal ratio

• Quantitative metrics to track:

  • Signal-to-noise ratio (SNR): Calculate as specific signal intensity divided by background

  • Minimum detectable concentration (MDC): Determine lowest amount of target producing distinguishable signal

  • Coefficient of variation (CV): Measure reproducibility across technical replicates

• Performance trending:

  • Create a quality control chart tracking SNR over time

  • Establish acceptance criteria (e.g., SNR decline <20% from original value)

  • Define action limits for antibody replacement or re-purification

Implementing these metrics allows for objective assessment of antibody stability and early detection of performance degradation .

How should Western blot data using SPAC13F5.07c antibody be quantified? (Basic)

Proper quantification of Western blot data using SPAC13F5.07c antibody involves several critical steps:

• Image acquisition:

  • Capture images within linear dynamic range of detection system

  • Avoid saturated pixels which prevent accurate quantification

  • Include a dilution series of standards if absolute quantification is needed

• Background correction methods:

  • Use rolling ball algorithm for uneven backgrounds

  • Employ local background subtraction for each lane

  • Apply identical correction parameters across all compared blots

• Normalization strategies:

  • Normalize to total protein (Ponceau S or SYPRO Ruby staining)

  • Use housekeeping proteins appropriate for yeast (e.g., actin, GAPDH)

  • Verify stability of reference proteins under your experimental conditions

• Statistical analysis:

  • Perform multiple independent experiments (n≥3)

  • Apply appropriate statistical tests based on data distribution

  • Report both normalized values and statistical significance

When presenting Western blot data, include both representative images and quantification graphs with error bars .

How can discrepancies between SPAC13F5.07c antibody results and other detection methods be reconciled? (Advanced)

When SPAC13F5.07c antibody results conflict with other detection methods, systematic reconciliation involves:

• Common discrepancy scenarios and resolution approaches:

• Orthogonal validation protocol:

  • Generate epitope-tagged SPAC13F5.07c constructs

  • Compare detection by antibody versus anti-tag antibodies

  • Use CRISPR-engineered strains with endogenous fluorescent tags

  • Compare microscopy results with antibody-based methods

• Benchmarking experiment:

  • Apply multiple detection methods to the same samples

  • Establish a detection threshold for each method

  • Create a correlation matrix to identify method-specific biases

These approaches help distinguish true biological findings from method-specific artifacts .

What considerations are important when using SPAC13F5.07c antibody for protein localization studies? (Advanced)

When conducting protein localization studies with SPAC13F5.07c antibody, several methodological considerations are critical:

• Fixation and permeabilization optimization:

  • Test multiple fixatives (4% paraformaldehyde, methanol, or glutaraldehyde)

  • Evaluate different permeabilization agents (0.1-0.5% Triton X-100, saponin, or digitonin)

  • Determine optimal fixation time to preserve structure while maintaining epitope accessibility

• Controls for specificity verification:

  • Include SPAC13F5.07c deletion strains as negative controls

  • Use competitive blocking with immunizing peptide

  • Compare localization with fluorescently-tagged SPAC13F5.07c

• Co-localization methodology:

  • Use established organelle markers appropriate for S. pombe

  • Apply proper statistical analysis (Pearson's correlation, Manders' coefficients)

  • Implement super-resolution techniques for precise localization

• Quantitative assessment:

  • Measure signal intensity across cellular compartments

  • Determine percentage of cells showing specific localization patterns

  • Track potential localization changes under different experimental conditions

These approaches ensure reliable and reproducible localization data while minimizing artifacts common in yeast immunofluorescence studies .

How can SPAC13F5.07c antibody be utilized in chromatin immunoprecipitation (ChIP) studies? (Advanced)

While not listed among validated applications, researchers may adapt SPAC13F5.07c antibody for ChIP studies with these methodological considerations:

• Antibody validation for ChIP:

  • Test epitope accessibility in crosslinked chromatin

  • Perform pilot ChIP-qPCR experiments with positive and negative genomic regions

  • Compare efficiency with different crosslinking methods (1% formaldehyde for 10-15 minutes is typical)

• Optimization protocol:

  • Test a range of antibody amounts (2-10 μg per ChIP reaction)

  • Evaluate various sonication conditions to achieve 200-500 bp fragments

  • Compare different washing stringencies to maximize signal-to-noise ratio

• Data analysis considerations:

  • Calculate percent input for quantification

  • Normalize to non-specific IgG control

  • Use spike-in controls for between-sample normalization

ChIP applications require rigorous controls due to the complex nature of chromatin and potential for non-specific interactions in the nuclear environment .

What approaches can integrate SPAC13F5.07c antibody with mass spectrometry for interactome studies? (Advanced)

Integrating SPAC13F5.07c antibody with mass spectrometry enables comprehensive interactome mapping through these methodological approaches:

• Immunoprecipitation-Mass Spectrometry (IP-MS) workflow:

  • Use mild lysis conditions to preserve protein complexes (e.g., 20 mM HEPES pH 7.4, 150 mM NaCl, 0.1% NP-40)

  • Crosslink antibody to beads to prevent antibody contamination in MS samples

  • Include stringent controls (IgG pulldowns, knockout strains)

  • Analyze data with SAINT, CRAPome, or similar statistical tools to filter contaminants

• Proximity-dependent labeling integration:

  • Generate SPAC13F5.07c fusions with BioID or TurboID

  • Validate fusion protein localization using the antibody

  • Compare antibody-based interactome with proximity labeling results

• Targeted versus discovery approaches:

  • Use parallel reaction monitoring (PRM) for targeted analysis of suspected interactors

  • Implement data-independent acquisition (DIA) for unbiased discovery

  • Create spectral libraries of potential interactors for improved identification

This integrated approach provides both validation of interactions and discovery of novel SPAC13F5.07c-associated proteins .

How does the use of SPAC13F5.07c antibody compare with CRISPR-based protein tagging approaches? (Advanced)

When deciding between antibody-based detection and CRISPR-based tagging of SPAC13F5.07c, consider these comparative aspects:

Complementary experimental design:

• Use CRISPR-tagged strains to validate antibody specificity

• Apply antibody-based detection to confirm tag doesn't affect localization

• Leverage antibody for biochemical assays and tagged protein for live imaging

• Combine approaches to distinguish between protein isoforms or modifications

This complementary strategy leverages strengths of both approaches while mitigating their respective limitations .

How do polyclonal SPAC13F5.07c antibodies compare with monoclonal alternatives? (Basic)

While the available SPAC13F5.07c antibody is polyclonal, researchers should understand the comparative advantages when considering future reagent development:

For SPAC13F5.07c research, polyclonal antibodies often provide advantages for initial characterization due to their robust signal and multiple epitope recognition, while monoclonal antibodies would offer benefits for standardized assays requiring consistent lot-to-lot performance .

What emerging technologies might enhance SPAC13F5.07c antibody applications? (Advanced)

Several emerging technologies hold promise for enhancing SPAC13F5.07c antibody applications:

• Single-molecule detection platforms:

  • DNA-PAINT super-resolution microscopy for precise localization

  • Single-molecule pull-down (SiMPull) for analyzing individual protein complexes

  • These approaches could reveal heterogeneity in SPAC13F5.07c interactions not detectable in bulk assays

• Microfluidic antibody applications:

  • Droplet-based single-cell Western blotting

  • Microfluidic immunoprecipitation with dramatically reduced sample requirements

  • These technologies enable analysis from limited samples or rare cell populations

• Spatial proteomics integration:

  • Combining antibody detection with mass spectrometry imaging

  • Multiplexed ion beam imaging (MIBI) for simultaneous detection of multiple targets

  • These methods provide spatial context to protein interactions and modifications

• Antibody engineering opportunities:

  • Single-chain variable fragments (scFvs) derived from the polyclonal population

  • Nanobody development against SPAC13F5.07c for improved penetration

  • These smaller binding reagents could access epitopes in complex structures

Implementing these technologies could significantly expand the utility of SPAC13F5.07c antibodies in both basic research and potential diagnostic applications .

What considerations are important when validating SPAC13F5.07c antibody across different yeast strains and growth conditions? (Advanced)

Comprehensive validation across strains and conditions is critical for robust SPAC13F5.07c antibody applications:

• Strain-specific validation protocol:

  • Test antibody performance in laboratory strains versus natural isolates

  • Validate detection in strains with varying SPAC13F5.07c sequence polymorphisms

  • Create calibration curves for quantification in different genetic backgrounds

• Growth condition considerations:

  • Examine epitope accessibility changes under stress conditions

  • Monitor post-translational modifications that may affect antibody binding

  • Establish detection limits across growth phases (log, stationary)

• Environmental factor assessment:

  • Temperature effects on protein expression and antibody affinity

  • Nutrient limitation impacts on target abundance

  • pH variation effects on epitope conformation

• Experimental design strategy:

  • Include strain-matched positive and negative controls

  • Implement spike-in standards for cross-condition normalization

  • Document condition-specific optimization parameters in standardized format

This systematic validation approach ensures reliable data interpretation across diverse experimental conditions and strain backgrounds, critical for comparative studies in yeast biology .