SPAC139.05 Antibody

What is SPAC139.05 and what type of antibody is used to detect it?

SPAC139.05 is a protein found in Schizosaccharomyces pombe (fission yeast). Antibodies against this target are typically used in research settings focused on yeast cellular processes and molecular biology . The commercially available SPAC139.05 Antibody offered by manufacturers like CUSABIO-WUHAN HUAMEI BIOTECH Co., Ltd. is designed specifically for research applications involving this fission yeast protein .

A typical SPAC139.05 Antibody application protocol would involve:

What validation methods should be used to confirm SPAC139.05 Antibody specificity?

Validation of antibody specificity is crucial for generating reliable research data. For SPAC139.05 Antibody, consider these methodological approaches:

• Knockout/knockdown controls: Compare antibody binding in wild-type vs. SPAC139.05 knockout or knockdown strains of S. pombe to verify specific target recognition .

• Western blot analysis: Confirm a single band of appropriate molecular weight. Multiple bands might indicate non-specific binding or protein degradation .

• Peptide competition assay: Pre-incubate the antibody with purified SPAC139.05 protein or peptide before application to samples. Diminished signal confirms specificity .

• Cross-species reactivity testing: Test the antibody against related proteins from other yeast species to evaluate potential cross-reactivity .

How should SPAC139.05 Antibody be stored for optimal performance?

Proper storage is essential for maintaining antibody activity. Based on standard protocols for research antibodies similar to SPAC139.05 Antibody:

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

• Aliquot upon first thaw to avoid repeated freeze-thaw cycles

• Working stocks can be maintained at 4°C for up to 2 weeks

• Many preparations contain 50% glycerol/PBS with preservatives like sodium azide for stability

How can SPAC139.05 Antibody be used in chromatin immunoprecipitation (ChIP) experiments?

While specific ChIP protocols for SPAC139.05 are not widely reported, methodological approaches based on similar yeast protein antibodies include:

• Crosslinking optimization: For S. pombe proteins, a 1% formaldehyde crosslinking for 15-20 minutes at room temperature is typically effective .

• Sonication parameters: Chromatin should be shared by sonication (e.g., 30s on/30s off at medium setting for approximately 10 minutes) to achieve fragments of 200-500 bp .

• Quantification methodology: ChIP signal values should be expressed as percentages of input DNA corrected for the no-antibody background to ensure accurate quantification .

• Controls: Include both positive controls (a known binding region) and negative controls (a non-binding region) to validate the specificity of immunoprecipitation .

What techniques can enhance signal detection when working with low abundance targets of SPAC139.05 Antibody?

Researchers working with low-abundance SPAC139.05 protein can employ several methodological enhancements:

• Signal amplification systems: Using tyramide signal amplification or polymeric detection systems can increase sensitivity by 10-50 fold .

• Optimized immunoprecipitation: For co-IP experiments, using specialized buffers with reduced detergent concentrations might preserve weaker protein-protein interactions .

• Super-resolution imaging techniques: For immunofluorescence applications, techniques like STORM or PALM can enhance detection of low-abundance proteins beyond standard confocal microscopy limits .

• Proximity ligation assays: When studying protein-protein interactions involving SPAC139.05, this technique can visualize interactions with higher sensitivity than conventional co-immunoprecipitation .

How can SPAC139.05 Antibody be integrated into multi-parameter flow cytometry experiments?

For researchers developing multi-parameter analysis protocols:

• Panel design considerations: When using SPAC139.05 Antibody in flow cytometry, it should be conjugated to fluorophores with minimal spectral overlap with other markers in your panel .

• Compensation matrix: Proper compensation is crucial when SPAC139.05 Antibody is labeled with fluorophores like APC or PE that have broad emission spectra .

• Optimizing fixation and permeabilization: Since SPAC139.05 is likely an intracellular target, fixation with 4% paraformaldehyde followed by permeabilization with 0.1% Triton X-100 is recommended .

• Fluorophore selection table:

What are the critical parameters for optimizing SPAC139.05 Antibody in Western blot applications?

Successful Western blotting with SPAC139.05 Antibody requires careful optimization:

• Sample preparation: For S. pombe proteins, effective extraction requires mechanical disruption (e.g., glass beads) combined with appropriate lysis buffers containing protease inhibitors .

• Blocking optimization: 5% non-fat dry milk in TBST is typically effective, but for phospho-specific applications, 5% BSA may yield better results .

• Primary antibody incubation: For optimal signal-to-noise ratio, incubation overnight at 4°C with gentle agitation is recommended .

• Secondary antibody selection: Choose species-appropriate secondary antibodies (typically anti-mouse or anti-rabbit) conjugated to HRP for standard chemiluminescent detection .

• Detection system: For quantitative analysis, digital imaging systems with appropriate dynamic range should be used rather than film-based detection .

What controls should be included when using SPAC139.05 Antibody in immunohistochemistry or immunofluorescence?

Proper controls are essential for result interpretation:

• Positive control: Include a sample known to express SPAC139.05 protein to verify the staining protocol is working correctly.

• Negative control: Use one of the following approaches:

  • Primary antibody omission

  • Isotype control antibody

  • Pre-adsorption with immunizing peptide

  • Samples from SPAC139.05 knockout organisms

• Subcellular localization verification: Compare observed localization with reported localization data for SPAC139.05 to ensure staining patterns are consistent with biological expectations.

• Cross-reactivity assessment: Test the antibody on tissues/cells not expected to express the target to identify potential non-specific binding .

How should epitope retrieval be optimized when using SPAC139.05 Antibody for fixed samples?

Epitope retrieval methods significantly impact antibody binding:

• Heat-induced epitope retrieval (HIER): Test multiple buffer systems:

  • Citrate buffer (pH 6.0)

  • EDTA buffer (pH 8.0-9.0)

  • Tris-EDTA (pH 9.0)

• Retrieval duration: Typically 15-20 minutes is sufficient, but optimization may be required for specific fixation conditions .

• Enzymatic retrieval: For some epitopes, enzymatic treatment with proteases like proteinase K may be more effective than heat-based methods.

• Fixation considerations: Antibody performance is directly related to fixation methods - shorter fixation times (4-24 hours) generally preserve epitopes better than extended fixation.

What are common causes of weak or absent signal when using SPAC139.05 Antibody?

When signals are weaker than expected, consider these methodological remedies:

• Antibody concentration: Titrate the antibody to determine optimal concentration; too low or too high concentrations can reduce specific signal .

• Target abundance: If SPAC139.05 is expressed at low levels, consider enrichment techniques or more sensitive detection methods .

• Epitope accessibility: The conformation of the target protein may obscure the epitope; try alternative fixation methods or epitope retrieval techniques .

• Antibody quality: Antibody effectiveness can diminish over time or with improper storage; use fresh aliquots and avoid repeated freeze-thaw cycles .

• Detection system sensitivity: Switch to more sensitive detection systems like tyramide signal amplification or polymeric HRP detection .

How can researchers validate novel binding partners or functions of SPAC139.05 using antibody-based approaches?

For researchers investigating new molecular interactions:

What approaches can be used to differentiate between phosphorylated and non-phosphorylated forms of SPAC139.05?

Post-translational modifications require specialized detection methods:

• Phospho-specific antibodies: Consider generating antibodies specific to known or predicted phosphorylation sites on SPAC139.05 .

• Lambda phosphatase treatment: Compare antibody binding before and after phosphatase treatment to determine phosphorylation-dependent binding .

• Phos-tag gels: These specialized acrylamide gels can separate phosphorylated from non-phosphorylated proteins based on mobility shifts.

• Mass spectrometry validation: For definitive identification of phosphorylation sites, MS/MS analysis following immunoprecipitation provides site-specific information .

• Mutation studies: Replacing predicted phosphorylation sites with alanine can confirm the importance of specific sites for antibody recognition.

What are emerging technologies that might enhance SPAC139.05 Antibody applications?

Forward-looking researchers should consider these evolving methodologies:

• Single-cell proteomics: Adapting antibody-based detection to single-cell resolution can reveal cell-to-cell variability in SPAC139.05 expression .

• Spatial proteomics: Techniques like Imaging Mass Cytometry or CODEX enable highly multiplexed antibody-based imaging for spatial context .

• Synthetic antibody technologies: Recombinant antibody fragments with enhanced specificity and reduced size might improve access to sterically hindered epitopes .

• Deep learning applications: AI-based image analysis can enhance detection and quantification of antibody staining, particularly in complex tissues or cellular contexts .

• In situ proximity ligation: This approach can visualize protein-protein interactions with higher sensitivity than conventional co-immunoprecipitation techniques .