SPAPB8E5.10 Antibody

What is SPAPB8E5.10 and why is it studied in research?

SPAPB8E5.10 is a sequence orphan gene in Schizosaccharomyces pombe (fission yeast) . As a sequence orphan, it lacks clear homology to known genes in other organisms, making it of interest for understanding S. pombe-specific biology. Research suggests some involvement in gene expression changes during stress responses, as it has been identified in studies of expression profiling of S. pombe acetyltransferase mutants .

Based on expression data analysis, SPAPB8E5.10 shows significant regulation in certain conditions:

The gene appears to be part of the transcriptional response in specific cellular contexts like cytoplasmic freezing adaptation .

How is SPAPB8E5.10 protein characterized functionally?

SPAPB8E5.10 remains functionally uncharacterized, but it is included in several gene lists associated with cellular processes in S. pombe . Current approaches to functional characterization include:

• Gene deletion studies using the genome-wide gene deletion library

• Expression profiling under various stress conditions

• Localization studies using fluorescent protein tagging

• Interaction studies with known pathway components

Antibodies against SPAPB8E5.10 are valuable tools for these characterization efforts, enabling protein detection in various experimental contexts.

How should I validate an SPAPB8E5.10 antibody for specificity in S. pombe research?

Proper antibody validation is crucial, particularly for relatively uncharacterized proteins like SPAPB8E5.10. A comprehensive validation approach should include :

• Genetic validation: Testing antibody reactivity in wild-type vs. SPAPB8E5.10 deletion strains. Absence of signal in the deletion strain confirms specificity.

• Western blot analysis: Verify that the antibody detects a protein of the expected molecular weight, with absence of significant non-specific bands.

• Immunoprecipitation followed by mass spectrometry: Confirm that the antibody enriches for SPAPB8E5.10 protein specifically. In one approach used for SpA5 antibodies, researchers "ultrasonically fragmented and centrifuged the bacterial fluid, took the supernatant and coincubated it with antibody overnight, then bound it with protein A beads the next day, and collected the eluate for mass spectrometry detection" .

• Orthogonal detection methods: Compare results with epitope-tagged versions of SPAPB8E5.10 detected via anti-tag antibodies.

The Antibody Society recommends that each antibody must be verified for the "precise application and tissue/cell type for which the antibody will be used, and all verification data must be reported openly" .

What epitope selection considerations are important for SPAPB8E5.10 antibody development?

For sequence orphans like SPAPB8E5.10, epitope selection requires careful consideration :

• Structural prediction: Use tools like AlphaFold2 to model the 3D theoretical structure of SPAPB8E5.10 and identify accessible regions.

• Sequence conservation: While SPAPB8E5.10 lacks clear homologs in other organisms, regions conserved among different S. pombe strains may represent functionally important domains.

• Post-translational modifications: Avoid regions likely to undergo phosphorylation, glycosylation, or other modifications that could interfere with antibody binding.

• Synthetic peptide validation: As demonstrated for other antibodies, synthesizing potential epitope peptides (e.g., 10-15 amino acids) and testing for binding can validate epitope selection before full antibody production .

An important lesson from other antibody studies is that "the specificity of an antibody is determined by the molecular characteristics of the Ig, and by those of the antigen, including the epitope's degree of folding/unfolding" .

What are the optimal conditions for using SPAPB8E5.10 antibodies in immunofluorescence of S. pombe cells?

For successful immunofluorescence in S. pombe using SPAPB8E5.10 antibodies, consider these methodological considerations:

• Cell wall digestion: S. pombe has a complex cell wall containing β-1,3-glucan, β-1,6-glucan, and α-1,3-glucan . Complete digestion is essential for antibody access.

  Protocol optimization:

  • Use zymolyase combined with lysing enzymes from Trichoderma

  • Monitor digestion by phase-contrast microscopy

  • Optimize digestion time (typically 30-60 minutes) to prevent overdigestion

• Fixation method: For membrane or cell wall-associated proteins, methanol fixation (-20°C, 6 minutes) often yields better results than formaldehyde.

• Antibody dilution: Start with 1:100 to 1:200 dilution for primary antibody incubation, similar to ranges used for other S. pombe proteins .

• Blocking solution: Use 5% BSA in PBS with 0.1% Triton X-100 to reduce non-specific binding.

• Validation controls: Include both wild-type and SPAPB8E5.10 deletion strains in each experiment to confirm specificity of observed signals.

How can I optimize Western blot protocols for SPAPB8E5.10 detection in S. pombe extracts?

Western blot optimization for SPAPB8E5.10 should address the particular challenges of fission yeast protein extraction:

• Cell lysis method: Mechanical disruption (e.g., bead beating) is essential for efficient S. pombe lysis due to its robust cell wall. Use acid-washed glass beads and perform 4-6 cycles of 30 seconds beating with cooling intervals.

• Sample preparation: Heat samples at 65°C rather than 95°C to prevent aggregation of membrane proteins (if SPAPB8E5.10 has membrane associations).

• Gel percentage: Use 10-12% gels based on the predicted molecular weight of SPAPB8E5.10.

• Transfer conditions: Semi-dry transfer at 15V for 30 minutes is often sufficient for most S. pombe proteins.

• Blocking and antibody incubation: 5% non-fat milk in TBST for blocking, with primary antibody incubation at 1:1000 dilution overnight at 4°C.

How can I investigate potential post-translational modifications of SPAPB8E5.10 using antibody-based approaches?

Studying post-translational modifications (PTMs) of SPAPB8E5.10 requires specialized antibody approaches:

• Phosphorylation analysis:

  • Use phospho-specific antibodies if phosphorylation sites are predicted

  • Compare standard Western blots with and without phosphatase treatment

  • Utilize Phos-tag SDS-PAGE to enhance separation of phosphorylated forms

  • Consider mass spectrometry analysis of immunoprecipitated protein for comprehensive phosphosite identification

• Glycosylation analysis:
  Based on studies with other S. pombe proteins , glycosylation analysis can be performed by:

  • Treatment with endoglycosidases prior to Western blotting

  • Comparison of protein migration in wild-type versus glycosylation mutant backgrounds

  • As demonstrated in a study of another S. pombe protein: "Sup11p:HA is hypo-mannosylated when expressed in an O-mannosylation mutant background"

• Ubiquitination detection:

  • Immunoprecipitate SPAPB8E5.10 under denaturing conditions

  • Probe with anti-ubiquitin antibodies

  • Use specific deubiquitinating enzyme inhibitors during cell lysis

This multi-faceted approach helps identify dynamic regulatory modifications affecting SPAPB8E5.10 function.

What approaches can resolve contradictory data when antibody-based localization conflicts with tagged protein studies?

When antibody-based localization results conflict with tagged protein approaches, consider these methodological solutions:

• Validate both approaches extensively:

  • Confirm specificity of the antibody using knockout controls

  • Verify functionality of tagged protein by complementation tests

  • Assess whether tagging interferes with localization signals

• Examine fixation artifacts:

  • Compare different fixation methods (methanol, formaldehyde, glutaraldehyde)

  • Use live-cell imaging with tagged proteins to eliminate fixation concerns

  • Consider that certain protein interactions may be disrupted during fixation

• Investigate epitope masking:

  • The antibody epitope may be masked in specific cellular compartments due to protein interactions

  • As noted in research, "The ability of the antibody to recognize the target protein is lost when the epitope is either masked or destroyed due to post-translational modifications"

• Cross-validate with proximity labeling:

  • Use BioID or APEX2 proximity labeling fused to SPAPB8E5.10

  • Identify neighboring proteins characteristic of specific cellular compartments

  • This approach can help resolve localization without relying solely on direct visualization

What are the most effective approaches for eliminating background when using SPAPB8E5.10 antibodies in S. pombe lysates?

High background is a common challenge when working with S. pombe lysates. Implement these methodological solutions:

• Pre-clear lysates:

  • Incubate lysates with Protein A/G beads before antibody addition

  • Add 1-5% of the host species normal serum to block non-specific binding sites

• Optimize blocking conditions:

  • Test different blocking agents (BSA, milk, fish gelatin, commercial blocking buffers)

  • Extend blocking time to 2 hours at room temperature

• Increase washing stringency:

  • Perform additional washes (5-6 times) with TBST or PBST

  • Include a high-salt wash (300-500 mM NaCl) to disrupt weak interactions

• Validate antibody specificity:

  • Test binding to lysates from SPAPB8E5.10 deletion strains

  • Pre-absorb antibody with recombinant SPAPB8E5.10 to confirm specific binding

• Reduce antibody concentration:

  • Titrate antibody to determine optimal concentration

  • As noted in studies, "a proper dilution of the antibody is required to preclude a highly specific antibody from binding to unrelated proteins at lower affinity"

How can I develop quantitative assays for SPAPB8E5.10 expression levels across different growth conditions?

Quantitative analysis of SPAPB8E5.10 requires careful assay development:

• Standardized Western blot quantification:

  • Include recombinant SPAPB8E5.10 protein standards on each blot

  • Use automated image analysis software for densitometry

  • Normalize to multiple loading controls (e.g., GAPDH, tubulin, total protein stain)

• ELISA development:

  • Establish a sandwich ELISA using two different SPAPB8E5.10 antibodies recognizing distinct epitopes

  • Generate a standard curve with purified recombinant protein

  • Protocol development would follow approaches similar to those used for other antibodies: "A suitable range of concentrations of this antibody for ELISA detection is 0.5-2 µg/mL. A standard curve consisting of doubling dilutions of the recombinant standard over the range of 4000 pg/mL - 30 pg/mL should be included in each ELISA plate"

• Flow cytometry for single-cell analysis:

  • Optimize cell permeabilization for intracellular staining

  • Use median fluorescence intensity for quantification

  • Include an isotype control antibody at the same concentration

• qPCR correlation:

  • Correlate protein levels with mRNA expression

  • Design specific primers for SPAPB8E5.10

  • Use this to validate antibody-based quantification methods

These quantitative approaches allow robust comparison of SPAPB8E5.10 expression across experimental conditions.