SPBC18H10.05 Antibody

How should I validate the specificity of an antibody against SPBC18H10.05?

Proper validation is critical before experimental use, as antibody performance varies significantly across applications. A multi-step validation approach should include:

• Western blot analysis comparing wild-type S. pombe with a SPBC18H10.05 deletion strain

• Immunoprecipitation followed by mass spectrometry to confirm the pulled-down protein

• Testing cross-reactivity with recombinant SPBC18H10.05 protein

• Epitope-tagging the endogenous protein and comparing antibody detection with tag-specific antibodies

This approach follows established validation protocols observed in multiple studies. For example, researchers studying Shb protein employed two positive controls: "The first one corresponds to an over-expression system resulting from HEK 293T cells transiently transfected with a cytomegalovirus (CMV) promoter-driven Stag-Shb-V5 construct. The second one corresponds to an endogenous expression situation where the HEK 293T-derived CE12 cell line has one of its Shb alleles V5-tagged" . Similar methodology should be applied to SPBC18H10.05 antibody validation.

What epitope selection considerations are important for SPBC18H10.05 antibody development?

When developing or selecting antibodies against S. pombe proteins like SPBC18H10.05, epitope selection significantly impacts specificity and functionality:

• Target unique regions that lack homology with other S. pombe proteins

• For polyclonal antibodies, N-terminal or C-terminal regions often make good targets

• Avoid transmembrane regions or highly conserved functional domains

• Consider protein modifications that might affect epitope accessibility

• For better immunogenicity, conjugate peptides to carrier proteins like keyhole limpet hemocyanin (KLH)

Commercial antibodies typically adopt these approaches, as seen with other proteins: "Anti-Shb: ab175553 - C-terminal amino acids (67-95) of mouse Shb conjugated to keyhole limpet haemocynin (KLH)" .

What are the recommended applications for SPBC18H10.05 antibodies in S. pombe research?

SPBC18H10.05 antibodies can be utilized in various experimental contexts, but researchers should note that a single antibody may not perform equally well across all applications:

• Western blotting for protein expression analysis

• Immunoprecipitation for protein interaction studies

• Chromatin immunoprecipitation (ChIP) if the protein has DNA-binding properties

• Immunofluorescence microscopy for subcellular localization

• Flow cytometry for quantitative analysis

As observed in antibody validation studies: "Several of the antibodies showed shortcomings or were not acceptable for detection of the endogenous protein. The few that could detect Shb were doing so in either western blotting or immunoprecipitation experiments but a given antibody could not work in both applications" . Therefore, validate each antibody specifically for your intended application.

What are the optimal conditions for immunoprecipitation experiments using SPBC18H10.05 antibodies?

For successful co-IP experiments with SPBC18H10.05 antibodies in S. pombe:

• Cell lysis: Test NP-40 (0.5-1%), RIPA, or milder detergents like digitonin

• Salt concentration: Start with 150mM NaCl, then adjust based on interaction strength

• Antibody amounts: Typically 2-5μg per 500μg of protein lysate

• Pre-clearing: Use protein A/G beads to remove non-specific binders

• Washing stringency: Balance between maintaining interactions and reducing background

• Controls: Include IgG control and when possible, a SPBC18H10.05 deletion strain

Effective approaches for S. pombe proteins include: "epitope-tagged each protein at the endogenous locus, affinity purified it from cell lysates, and identified co-precipitating proteins by liquid chromatography - tandem mass spectrometry" .

How can SPBC18H10.05 antibodies be used to study protein complex formation?

For comprehensive identification of SPBC18H10.05-interacting partners:

• Buffer optimization: Test various ionic strengths and detergent concentrations

• Cross-linking approaches: Consider formaldehyde or DSP cross-linking to capture transient interactions

• Mass spectrometry analysis: Use both label-free and isotope-labeled approaches for quantitative assessment

• Validation: Confirm key interactions by reciprocal IP or proximity ligation assays

This approach has proven successful in studying fission yeast protein complexes: "Saf5 was found to specifically associate with components of the splicing machinery, most notably, all components of the core snRNP including Smd3" .

How can ChIP protocols be optimized using SPBC18H10.05 antibodies for chromatin studies?

Chromatin immunoprecipitation with SPBC18H10.05 antibodies requires specific optimization for fission yeast:

• Cell fixation: Use 1% formaldehyde for 15-30 minutes at room temperature

• Cell lysis: Optimize mechanical disruption with glass beads for S. pombe's tough cell wall

• Sonication: Adjust conditions to achieve chromatin fragments of 200-500bp

• Antibody concentration: Titrate to determine optimal amount (typically 2-5μg per ChIP reaction)

• Controls: Include IgG control, input samples, and a strain with deleted or tagged SPBC18H10.05

For validation of ChIP results, consider orthogonal approaches as used in centromeric studies, where researchers measured "centromeric siRNAs in all the mutants, indicative of defective RNAi-mediated processing of non-coding centromeric transcripts" .

How can conflicting results between different SPBC18H10.05 antibody lots be reconciled?

When facing inconsistent results between antibody lots:

• Epitope mapping: Determine if different lots recognize distinct epitopes

• Validation series: Re-validate each lot using knockout/knockdown controls

• Sensitivity testing: Compare detection limits using dilution series of recombinant protein

• Cross-reactivity assessment: Test against related proteins or in knockout systems

Researchers have noted significant variability: "The few that could detect Shb were doing so in either western blotting or immunoprecipitation experiments but a given antibody could not work in both applications" . This highlights the importance of comprehensive validation for each experimental application.

What are the recommended protocols for generating and validating knockout controls?

For creating effective knockout controls in S. pombe:

• CRISPR-Cas9 approach: Design guide RNAs targeting early exons of SPBC18H10.05

• Homologous recombination: Use antibiotic resistance cassettes flanked by homology regions

• Verification methods: Combine PCR genotyping, sequencing, RT-qPCR, and western blotting

• Phenotypic characterization: Assess growth, morphology, and related pathway functions

Systematic gene deletion approaches have been successfully employed in fission yeast studies to create comprehensive mutant collections: "we obtained a comprehensive catalog of autophagy genes in this highly tractable organism, including genes encoding three heretofore unidentified core Atg proteins" .

Antibody Specifications and Applications Table

What approaches can be used to study post-translational modifications of SPBC18H10.05?

To investigate PTMs of SPBC18H10.05:

• Modification-specific antibodies: Consider developing phospho-, acetyl-, or ubiquitin-specific antibodies

• Enrichment strategies: Use phosphatase inhibitors, deacetylase inhibitors during sample preparation

• Sequential immunoprecipitation: First IP with SPBC18H10.05 antibody, then probe with modification-specific antibodies

• Mass spectrometry validation: Use IP followed by MS to map modification sites

These approaches are particularly important for studying proteins involved in signaling pathways, as demonstrated in studies of platform proteins "involved in receptor tyrosine kinase signalling" .

How can SPBC18H10.05 antibodies be used to investigate protein dynamics during the cell cycle?

For cell-cycle dependent studies in S. pombe:

• Synchronization methods: Optimize nitrogen starvation/release for S. pombe

• Time-course sampling: Collect samples at regular intervals following synchronization

• Fixation protocols: Optimize to preserve cell cycle stage and protein localization

• Quantitative imaging: Use fluorescence intensity measurements for relative protein levels

• Co-localization studies: Combine with established cell cycle markers

Similar approaches have been used effectively to study protein dynamics in fission yeast, allowing researchers to "systematically examined the subcellular localization of fission yeast autophagy factors" .

What are the considerations for using SPBC18H10.05 antibodies in multi-protein complex studies?

When investigating complex assemblies:

• Buffer optimization: Test various ionic strengths and detergent concentrations

• Sequential immunoprecipitation: Consider tandem IP to isolate specific subcomplexes

• Density gradient fractionation: Combine with antibody detection to identify complex size

• Cross-linking strategies: Use graduated cross-linking to preserve interactions

As demonstrated in fission yeast studies: "Saf5 was found to specifically associate with components of the splicing machinery, most notably, all components of the core snRNP including Smd3" , revealing how proper immunoprecipitation conditions can uncover complex protein assemblies.

How can SPBC18H10.05 antibodies be effectively used in chromatin fractionation experiments?

For chromatin association studies in fission yeast:

• Fractionation protocols: Optimize cytoplasmic, nucleoplasmic, and chromatin fraction separation

• Salt extraction series: Use increasing NaCl concentrations (100mM to 500mM) to determine binding strength

• Nuclease treatment: Apply DNase or RNase to distinguish DNA vs. RNA-mediated associations

• Controls: Include histone antibodies (chromatin), tubulin (cytoplasmic), and nuclear markers

These approaches have been valuable in characterizing novel factors in fission yeast. For instance, researchers identified that "Ctl1 interacts with Atg9 and is required for autophagosome formation" using similar biochemical fractionation approaches.