SPAC22E12.18 Antibody

What is SPAC22E12.18 and why is it significant in S. pombe research?

SPAC22E12.18 is a conserved fungal protein in Schizosaccharomyces pombe (fission yeast) that has been identified as a protein-coding gene with specific subcellular localization. According to BioGRID data, SPAC22E12.18 has two confirmed Gene Ontology Cellular Component annotations: cytoplasm (ISO) and cytosol (IDA) . The protein has a UniProt ID of Q10368 and represents a valuable target for studying conserved fungal cellular processes.

The significance of this protein lies in its conservation across fungal species, indicating potential evolutionary importance. Research using antibodies against this protein can help elucidate its function in fundamental cellular processes in S. pombe, which often serves as a model organism for understanding eukaryotic cell biology.

What applications are validated for SPAC22E12.18 antibody?

Based on available data, SPAC22E12.18 antibody can be used in multiple experimental applications common to fungal protein research:

When using this antibody, researchers should note that validation techniques similar to those used for other S. pombe proteins may apply. For instance, experimental approaches for membrane preparation from S. pombe and spheroblasting techniques as described in methodological literature would be applicable.

What protocols are recommended for protein extraction when using SPAC22E12.18 antibody?

For optimal results with SPAC22E12.18 antibody, proper protein extraction from S. pombe is critical. Based on established protocols for similar fission yeast proteins:

Membrane Preparation Protocol:

• Harvest S. pombe cells at mid-log phase

• Resuspend cells in extraction buffer containing protease inhibitors

• Disrupt cells using glass beads or enzymatic methods

• Centrifuge at low speed to remove cell debris

• Ultracentrifuge supernatant to isolate membrane fractions

• Resuspend pellet in appropriate buffer for downstream applications

For spheroblasting of S. pombe (essential for certain applications):

• Treat cells with zymolyase or lysing enzymes in buffer containing sorbitol

• Monitor spheroblast formation microscopically

• Gently centrifuge to collect spheroblasts

• Lyse spheroblasts using gentle detergents

These protocols are based on established methods for S. pombe protein extraction as referenced in methodological research .

How can I validate the specificity of SPAC22E12.18 antibody in my experiments?

Antibody validation is crucial for ensuring experimental rigor. For SPAC22E12.18 antibody, consider implementing these validation strategies:

Primary Validation Methods:

• Genetic Knockouts/Knockdowns: Generate a conditional nmt81-SPAC22E12.18 mutant similar to approaches used for other S. pombe proteins . The absence of signal in Western blot or immunofluorescence experiments would confirm specificity.

• Epitope Tagging: Create a fusion protein (SPAC22E12.18-HA or SPAC22E12.18-GFP) and perform co-localization studies with the antibody. Signals should overlap to demonstrate specificity.

• Mass Spectrometry: Perform immunoprecipitation followed by mass spectrometry analysis to confirm that the pulled-down protein is indeed SPAC22E12.18.

Advanced Validation:

• Preabsorption tests using recombinant SPAC22E12.18 protein

• Cross-species validation in closely related fungi

• Peptide competition assays

Researchers should note that proper validation experiments are critical, especially since SPAC22E12.18 is a conserved fungal protein that may share homology with other proteins.

What is the optimal immunofluorescence protocol for subcellular localization of SPAC22E12.18?

For precise subcellular localization of SPAC22E12.18 in S. pombe cells, the following optimized protocol is recommended:

Methanol Fixation and Immunofluorescence Protocol:

• Harvest cells at mid-log phase

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

• Wash 3× with PEM buffer (100 mM PIPES, 1 mM EGTA, 1 mM MgSO₄, pH 6.9)

• Permeabilize with 1% Triton X-100 in PEM for 2 minutes

• Block with 5% BSA in PEMBAL for 60 minutes

• Incubate with primary SPAC22E12.18 antibody (1:100-1:500 dilution) overnight at 4°C

• Wash 3× with PEMBAL

• Incubate with fluorophore-conjugated secondary antibody for 2 hours at room temperature

• Wash 3× with PEMBAL

• Mount slides with antifade reagent containing DAPI

This protocol is based on established methods for immunofluorescence in S. pombe . Given SPAC22E12.18's localization to the cytoplasm and cytosol , researchers should observe diffuse cytoplasmic staining.

For co-localization studies, researchers may use markers for specific organelles to further characterize the protein's distribution.

How can I optimize SPAC22E12.18 antibody for single-cell analysis techniques?

Single-cell analysis techniques are becoming increasingly important in understanding cell-to-cell variability. For SPAC22E12.18 antibody applications in single-cell techniques:

For Flow Cytometry:
The antibody can be adapted for flow cytometry applications similar to those established for human CD18 antibody , which has been successfully used in single-cell applications:

For Single-Cell RNA-seq + Protein Detection:

• Follow established protocols for cell preparation from yeast cultures

• Use oligo-conjugated SPAC22E12.18 antibody (similar to the 5CFLX conjugation approach )

• Implement a barcode sequence for antibody identification

• Process using 10x Genomics or similar platforms

These recommendations are based on techniques successfully applied to other proteins in single-cell analysis contexts .

What strategies can address potential cross-reactivity with other conserved fungal proteins?

Cross-reactivity is a significant concern when working with conserved fungal proteins. For SPAC22E12.18 antibody:

Analytical Approaches:

• Sequence alignment analysis: Compare SPAC22E12.18 with homologous proteins in other fungi to identify unique epitopes

• Western blot analysis: Test antibody against lysates from related fungal species

• Immunoprecipitation-mass spectrometry: Identify all proteins pulled down by the antibody

Experimental Solutions:

• Pre-absorption with recombinant homologous proteins: Eliminate cross-reactivity by pre-incubating antibody with potential cross-reactive proteins

• Epitope-specific antibody design: Commission custom antibodies against unique regions of SPAC22E12.18

• CRISPR-based epitope tagging: Tag endogenous SPAC22E12.18 and use tag-specific antibodies

For researchers studying evolutionary aspects, it's worth noting that this protein is conserved across fungal species, and careful validation is needed when extending studies beyond S. pombe.

How can I troubleshoot weak or inconsistent signals when using SPAC22E12.18 antibody?

Weak or inconsistent signals are common challenges in antibody-based experiments. For SPAC22E12.18 antibody:

Systematic Troubleshooting Approach:

Advanced Solutions:

• Signal amplification using tyramide signal amplification (TSA)

• Alternative detection methods (e.g., proximity ligation assay)

• Epitope retrieval techniques for fixed samples

Remember that SPAC22E12.18 is localized to the cytoplasm and cytosol , which should guide your expectations for localization patterns.

What approaches can detect potential protein-protein interactions involving SPAC22E12.18?

Understanding the protein interaction network of SPAC22E12.18 can provide insights into its function. Based on BioGRID data, SPAC22E12.18 has 3 reported interactors and 3 interactions .

Recommended Methodologies:

• Co-immunoprecipitation (Co-IP):

  • Use SPAC22E12.18 antibody for immunoprecipitation

  • Analyze co-precipitated proteins by Western blot or mass spectrometry

  • Control experiments should include IgG control and validation in knockout strains

• Proximity-dependent Biotin Identification (BioID):

  • Create a fusion of SPAC22E12.18 with BioID2 biotin ligase

  • Express in S. pombe and analyze biotinylated proteins

  • Compare results with control BioID2 fusions

• Yeast Two-Hybrid Screening:

  • Use SPAC22E12.18 as bait to screen S. pombe cDNA library

  • Validate interactions with targeted Y2H and in vivo methods

  • Consider split-ubiquitin Y2H for membrane-associated interactions

These approaches can help expand our understanding of SPAC22E12.18's functional role in S. pombe cellular processes.

How can I investigate potential post-translational modifications of SPAC22E12.18?

Post-translational modifications (PTMs) often regulate protein function. For investigating PTMs of SPAC22E12.18:

Experimental Strategy:

• Immunoprecipitation and Mass Spectrometry:

  • Immunoprecipitate SPAC22E12.18 using specific antibody

  • Digest purified protein and analyze by LC-MS/MS

  • Use specialized search algorithms to identify PTMs

• Western Blot Analysis:

  • Use PTM-specific antibodies (phospho, acetyl, ubiquitin, etc.)

  • Employ mobility shift assays to detect modifications

  • Compare PTM status under different conditions

• Site-directed Mutagenesis:

  • Identify putative modification sites via bioinformatics

  • Create point mutations at these sites

  • Assess functional consequences in vivo

These approaches can reveal important regulatory mechanisms for SPAC22E12.18 function that go beyond simple protein expression levels.