SPAC24H6.08 Antibody

What is SPAC24H6.08 protein and why is it significant in Schizosaccharomyces pombe research?

SPAC24H6.08 (UniProt: Q09762) is a protein expressed in the fission yeast Schizosaccharomyces pombe (strain 972 / ATCC 24843). This protein is of interest in studying fundamental cellular processes in S. pombe, which serves as an important model organism for understanding eukaryotic cell biology. Research involving SPAC24H6.08 contributes to our understanding of cellular mechanisms that may be conserved across species. The antibody against this protein enables researchers to detect, quantify, and isolate the protein for various experimental applications, providing insights into its function and regulation within cellular pathways.

What applications has the SPAC24H6.08 Antibody been validated for?

The SPAC24H6.08 Antibody has been specifically validated for Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blot (WB) applications . These techniques allow researchers to detect and quantify the presence of SPAC24H6.08 protein in various sample types. While these are the validated applications, researchers may optimize protocols for additional applications such as immunoprecipitation, immunohistochemistry, or immunofluorescence, though such applications would require extensive validation before reliable data can be obtained.

What are the optimal storage conditions for SPAC24H6.08 Antibody?

For optimal preservation of antibody activity, SPAC24H6.08 Antibody should be stored at -20°C or -80°C upon receipt . Repeated freeze-thaw cycles should be avoided as they can lead to denaturation of the antibody and loss of activity. The antibody is supplied in a storage buffer consisting of 50% glycerol, 0.01M PBS at pH 7.4, with 0.03% Proclin 300 as a preservative . The high glycerol content helps prevent freezing damage, allowing for aliquoting and long-term storage. For working solutions, store at 4°C for up to one week, but return to -20°C or -80°C for longer storage periods.

How can I optimize Western blot protocols for SPAC24H6.08 Antibody?

Optimizing Western blot protocols for SPAC24H6.08 Antibody requires systematic adjustment of several parameters:

Sample Preparation:

• Ensure complete cell lysis using methods appropriate for S. pombe, such as glass bead disruption or enzymatic digestion followed by detergent treatment

• Include protease inhibitors to prevent degradation of the target protein

• Determine optimal protein loading (typically 10-50 μg of total protein per lane)

Antibody Conditions:

• Start with a 1:1000 dilution of the antibody and adjust based on signal intensity

• Optimize blocking conditions (typically 5% non-fat milk or BSA in TBST)

• Test different incubation times and temperatures (overnight at 4°C often yields best results)

Detection:

• Choose appropriate secondary antibody (anti-rabbit IgG)

• Optimize exposure times based on signal-to-noise ratio

• Consider enhanced chemiluminescence (ECL) or fluorescence-based detection systems

Similar to standard antibody optimization processes used in studies of human antibodies, these parameters need to be adjusted systematically while maintaining appropriate controls .

What controls should be included when working with SPAC24H6.08 Antibody in ELISA?

For rigorous ELISA experiments with SPAC24H6.08 Antibody, include these essential controls:

Positive Controls:

• Purified recombinant SPAC24H6.08 protein at known concentrations

• Lysate from wild-type S. pombe expressing SPAC24H6.08

Negative Controls:

• Lysate from SPAC24H6.08 deletion mutant (if available)

• Lysate from unrelated yeast species (to assess cross-reactivity)

Technical Controls:

• No primary antibody control (to assess secondary antibody non-specific binding)

• No antigen control (to establish background signal)

• Isotype control (rabbit IgG at the same concentration)

Standard Curve:
Establish a standard curve using purified recombinant protein to enable quantification

This multi-level control strategy ensures reliable data interpretation and is similar to approaches used in other antibody-based studies .

How can I validate the specificity of SPAC24H6.08 Antibody for my specific S. pombe strain?

To validate specificity for your specific S. pombe strain, implement these approaches:

• Genetic validation: Test the antibody on samples from wild-type and SPAC24H6.08 knockout strains. Absence of signal in the knockout confirms specificity.

• Epitope blocking: Pre-incubate the antibody with excess purified recombinant SPAC24H6.08 protein before use in your assay. Reduction or elimination of signal indicates specificity.

• Expression correlation: Compare protein detection levels with mRNA expression data across different conditions or strains.

• Mass spectrometry validation: Perform immunoprecipitation followed by mass spectrometry to confirm the identity of the captured protein.

• Signal depletion: Perform successive immunoprecipitations to deplete the protein and demonstrate corresponding signal reduction.

These validation strategies provide complementary evidence for antibody specificity and are particularly important when working with different genetic backgrounds or experimental conditions .

What sample preparation methods are recommended for detecting SPAC24H6.08 in S. pombe?

Effective sample preparation is critical for detecting SPAC24H6.08 protein in S. pombe samples:

For Western Blot Analysis:

• Harvest cells during mid-log phase (OD600 of 0.5-0.8)

• Wash cells with cold PBS or TBS to remove media components

• Disrupt the cell wall using one of these methods:

  • Mechanical disruption with glass beads (0.5mm) in a bead beater

  • Enzymatic digestion with zymolyase followed by detergent lysis

• Include protease inhibitor cocktail and phosphatase inhibitors if phosphorylation states are relevant

• Clear lysate by centrifugation (14,000 × g for 15 minutes at 4°C)

• Determine protein concentration using Bradford or BCA assay

• Denature samples in Laemmli buffer at 95°C for 5 minutes

For ELISA:

• Follow similar cell disruption methods as for Western blot

• After clearing the lysate, dilute in carbonate/bicarbonate buffer (pH 9.6) for coating

• Coat plates with 1-10 μg/mL of total protein

• Alternatively, perform a sandwich ELISA using a capture antibody against another epitope

These preparation methods ensure optimal protein extraction while preserving epitope integrity for antibody recognition .

How can I assess cross-reactivity of SPAC24H6.08 Antibody with proteins from related yeast species?

Assessing cross-reactivity with related yeast species requires a systematic approach:

• Sequence homology analysis: Perform bioinformatic analysis to identify homologous proteins in related species using tools like BLAST or Clustal Omega. Calculate sequence identity percentages to predict potential cross-reactivity.

• Western blot analysis: Prepare protein extracts from multiple yeast species (e.g., S. cerevisiae, S. japonicus, S. octosporus) using identical protocols. Run samples alongside S. pombe extracts and probe with SPAC24H6.08 Antibody.

• Epitope mapping: If possible, identify the specific epitopes recognized by the polyclonal antibody through techniques such as peptide arrays or phage display. Compare these epitope sequences across species.

• Competitive ELISA: Perform competitive binding assays using purified homologous proteins from related species to quantify relative binding affinities.

• Immunoprecipitation followed by mass spectrometry: Use the antibody to immunoprecipitate proteins from lysates of related species, then identify pulled-down proteins by mass spectrometry to detect cross-reactive proteins.

This comprehensive approach allows for quantitative assessment of cross-reactivity, which is important for interpreting experimental results involving multiple yeast species .

What approaches can be used to determine the epitope(s) recognized by SPAC24H6.08 Antibody?

Determining the epitopes recognized by this polyclonal antibody can be achieved through several complementary techniques:

• Peptide array analysis: Synthesize overlapping peptides (typically 15-20 amino acids with 5-10 amino acid overlaps) spanning the entire SPAC24H6.08 protein sequence. Screen the peptide array with the antibody to identify reactive peptides.

• Deletion mutant analysis: Create truncated versions of the SPAC24H6.08 protein and test antibody reactivity against each fragment to narrow down the binding region.

• Site-directed mutagenesis: Once candidate regions are identified, introduce point mutations to identify specific amino acids critical for antibody binding.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Compare exchange patterns of the protein alone versus antibody-bound protein to identify protected regions that likely represent epitopes.

• X-ray crystallography or cryo-EM: For definitive epitope mapping, solve the structure of the antibody-antigen complex, though this approach is resource-intensive.

Understanding the specific epitopes recognized by the antibody provides valuable insight into its performance characteristics and potential limitations in different applications .

How can I use SPAC24H6.08 Antibody in co-immunoprecipitation experiments to identify interaction partners?

To effectively use SPAC24H6.08 Antibody for co-immunoprecipitation (co-IP) of protein interaction partners:

• Crosslinking optimization: Test different crosslinking conditions (if using crosslinking) with formaldehyde (0.1-1%) or DSP (1-2 mM) to preserve transient interactions.

• Lysis buffer selection: Choose a lysis buffer that maintains protein-protein interactions while allowing effective extraction:

  • Start with a gentle buffer (e.g., 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40 or 0.5% Triton X-100)

  • Adjust salt concentration (150-300 mM) and detergent type/concentration based on interaction strength

• Antibody coupling: Immobilize the SPAC24H6.08 Antibody to protein A/G beads, or consider using direct covalent coupling to reduce antibody contamination in the eluate.

• Pre-clearing: Pre-clear lysates with protein A/G beads to reduce non-specific binding.

• Controls: Include essential controls:

  • IgG isotype control

  • Input sample

  • SPAC24H6.08 knockout or knockdown (negative control)

• Elution strategy: Use either low pH, high pH, or competitive elution with excess antigen/peptide.

• Analysis: Analyze co-IP products by mass spectrometry (preferably quantitative approaches like SILAC or TMT) to identify specific interaction partners compared to controls .

This approach allows for the identification of protein complexes associated with SPAC24H6.08 in S. pombe, providing insights into its cellular functions.

What quantitative methods can I use to analyze SPAC24H6.08 expression levels across different experimental conditions?

Several quantitative methods can reliably measure SPAC24H6.08 expression levels:

Western Blot Quantification:

• Use a dilution series of recombinant SPAC24H6.08 protein to create a standard curve

• Ensure samples fall within the linear range of detection

• Normalize to loading controls (e.g., actin, tubulin)

• Use image analysis software for densitometry (e.g., ImageJ, Image Lab)

Quantitative ELISA:

• Develop a sandwich ELISA using SPAC24H6.08 Antibody as either capture or detection antibody

• Generate a standard curve using purified recombinant protein

• Analyze samples in technical triplicates to assess variability

Quantitative Proteomics:

• Use SILAC (Stable Isotope Labeling with Amino acids in Cell culture) to label proteins

• Immunoprecipitate SPAC24H6.08 from mixed lysates

• Analyze by mass spectrometry to determine relative abundance ratios

• Consider targeted approaches like Selected Reaction Monitoring (SRM) for highest sensitivity

Capillary Western (Wes):

• Automated capillary-based immunoassay for higher sensitivity and reproducibility

• Requires smaller sample volumes than traditional Western blot

• Provides broader linear dynamic range for quantification

Each method offers different advantages in terms of sensitivity, throughput, and specificity. Selection should be based on experimental requirements and available equipment .

How should I troubleshoot weak or absent signal when using SPAC24H6.08 Antibody?

When encountering weak or absent signals with SPAC24H6.08 Antibody, investigate these common causes systematically:

Sample-Related Issues:

• Low target protein expression: Verify expression using alternative methods (RT-PCR, RNA-seq)

• Protein degradation: Add fresh protease inhibitors, reduce sample processing time

• Inefficient extraction: Test alternative lysis methods for S. pombe (e.g., stronger mechanical disruption)

Antibody-Related Issues:

• Antibody degradation: Check storage conditions, avoid repeated freeze-thaw cycles

• Suboptimal concentration: Test a concentration series (1:100 to 1:5000)

• Epitope masking: Try different sample preparation methods to expose epitopes (e.g., different detergents)

Protocol-Related Issues:

• Inefficient protein transfer: Verify transfer efficiency with reversible protein stain

• Excessive blocking: Reduce blocking time or concentration

• Insufficient incubation time: Extend primary antibody incubation (overnight at 4°C)

• Detection system issues: Test alternative secondary antibodies or detection reagents

Experiment-Specific Issues:

• Create a positive control: Overexpress SPAC24H6.08 in S. pombe

• Verify antibody functionality: Test with recombinant SPAC24H6.08 protein

This structured troubleshooting approach helps identify and resolve specific issues affecting antibody performance .

What are the recommended fixation methods for immunofluorescence studies with SPAC24H6.08 Antibody?

Although SPAC24H6.08 Antibody has not been specifically validated for immunofluorescence, researchers may optimize protocols using these fixation approaches for S. pombe:

Formaldehyde Fixation:

• Harvest cells in mid-log phase

• Fix with 3-4% formaldehyde for 30 minutes at room temperature

• Wash 3× with PBS

• Digest cell wall with zymolyase (1 mg/ml) for 30-60 minutes at 37°C

• Permeabilize with 0.1% Triton X-100 for 5 minutes

Methanol Fixation:

• Harvest cells as above

• Fix with cold methanol (-20°C) for 6-8 minutes

• Wash with PBS

• No additional permeabilization required

Comparison of Fixation Methods for S. pombe Immunofluorescence:

Test each method to determine which best preserves the epitope recognized by SPAC24H6.08 Antibody while maintaining cellular morphology.

How can I determine the optimal antibody concentration for my specific application?

Determining the optimal antibody concentration requires systematic titration:

• For Western blot:

  • Prepare a single blot with identical protein samples

  • Cut the membrane into strips

  • Test a range of primary antibody dilutions (1:250, 1:500, 1:1000, 1:2000, 1:5000)

  • Keep all other parameters constant

  • Select the dilution that provides the best signal-to-noise ratio

• For ELISA:

  • Prepare a checkerboard titration:

    • Coat plates with a range of antigen concentrations (columns)

    • Test a range of antibody dilutions (rows)

  • Generate a series of curves to identify the optimal combination

  • The optimal concentration should provide a good dynamic range while minimizing background

• For immunoprecipitation:

  • Test 1, 2, 5, and 10 μg of antibody per mg of total protein

  • Analyze pull-down efficiency by Western blot

  • Select the lowest antibody concentration that achieves maximum target recovery

Document the optimization process for future reference and reproducibility. The optimal concentration may vary between antibody lots and sample types .

What strategies can be employed to reduce background when working with SPAC24H6.08 Antibody?

Managing background signal is crucial for obtaining clean, interpretable results:

For Western Blot:

• Optimize blocking: Test different blocking agents (5% milk, 3% BSA, commercial blockers) and times

• Increase washing stringency: Use 0.1-0.3% Tween-20 in TBS and increase wash time/frequency

• Dilute antibody in fresh blocking buffer

• Pre-adsorb antibody with non-specific proteins (e.g., acetone powder from non-expressing cells)

• Optimize secondary antibody concentration (typically 1:5000-1:20000)

For ELISA:

• Use highly purified blocking proteins to reduce non-specific binding

• Include 0.05% Tween-20 in all buffers after blocking

• Consider adding low concentrations (0.1-0.5%) of irrelevant species serum

• Optimize plate washing procedures (number of washes, volume, washing technique)

For Immunoprecipitation:

• Pre-clear lysates with Protein A/G beads before adding antibody

• Use more stringent wash buffers for final washes

• Consider cross-linking antibody to beads to prevent antibody leaching

• Include competing proteins (e.g., BSA) in wash buffers at low concentrations

General Strategies:

• Filter all buffers (0.22 μm) to remove particulates

• Use highly purified water for all solutions

• Prepare fresh buffers regularly

• Handle samples and reagents with clean technique to avoid contamination

These strategies can significantly improve signal-to-noise ratio across various applications .