SPAC6F6.04c Antibody

What is SPAC6F6.04c and what organism is it derived from?

SPAC6F6.04c is an uncharacterized membrane protein found in Schizosaccharomyces pombe (strain 972 / ATCC 24843), commonly known as fission yeast. This protein is identified by UniProt accession number O14237 and is the target of specific antibodies used in various research applications . The gene coding for this protein is part of the systematic nomenclature used for S. pombe, where "SPAC" denotes chromosome I location, followed by cosmid number (6F6) and the specific open reading frame (04c).

The ".04c" designation indicates it is the fourth gene on the cosmid, with "c" signifying that it is transcribed from the complementary DNA strand. Understanding the nature of this protein is essential for researchers designing experiments targeting membrane-associated processes in fission yeast.

What forms of SPAC6F6.04c antibodies are commercially available?

Several forms of SPAC6F6.04c antibodies are available to researchers, each with specific applications and characteristics. The primary antibody (CSB-PA517646XA01SXV) is available in different sizes (2ml/0.1ml), designed for research applications like Western blotting and ELISA . Additionally, the recombinant protein itself is available in multiple expression systems:

These options allow researchers to select the most appropriate form based on their specific experimental requirements and the biological questions being addressed .

What are the validated applications for SPAC6F6.04c antibodies?

Based on available data, SPAC6F6.04c antibodies have been validated for several research applications including ELISA and Western blotting (WB) for protein identification . These antibodies can be utilized in fundamental research applications exploring membrane protein function in S. pombe, particularly when investigating cellular processes such as heterochromatin maintenance, which has been studied extensively in this model organism .

The antibodies may also be suitable for immunofluorescence studies, although specific protocols would need to be optimized based on general principles of recombinant antibody applications in microscopy. As with all research antibodies, validation in the specific experimental context is essential for reliable results.

How should researchers select between different expression systems for SPAC6F6.04c antibodies?

The selection of an appropriate expression system depends primarily on the research application and the specific protein characteristics required. When designing experiments with SPAC6F6.04c antibodies, consider the following criteria:

• Native conformation requirements: If studying interactions that depend on authentic post-translational modifications, yeast (CSB-YP517646SXV) or mammalian (CSB-MP517646SXV) expression systems would be optimal as they provide more native-like modifications .

• Detection system compatibility: For applications requiring biotinylated antibodies (such as certain flow cytometry or pull-down protocols), the Avi-tag biotinylated version (CSB-EP517646SXV-B) offers specific advantages through the biotin-streptavidin interaction system .

• Scale and economic considerations: For large-scale preliminary studies, E. coli-expressed proteins (CSB-EP517646SXV) typically provide higher yields at lower costs .

• Experimental technique: Flow cytometry applications benefit particularly from recombinant antibodies due to their enhanced specificity and batch-to-batch consistency, which is crucial for quantitative analyses .

Researchers should match the expression system to their specific experimental requirements, considering that each system offers different advantages in terms of protein folding, modification, and functionality.

What are the optimal validation methods for SPAC6F6.04c antibodies?

Rigorous validation of SPAC6F6.04c antibodies is essential for reliable research outcomes. The recommended validation approach should include:

• Specificity testing: Compare binding in wild-type cells versus genetic knockout/knockdown models of SPAC6F6.04c. This is particularly important in S. pombe studies where genetic manipulation is well-established .

• Cross-reactivity assessment: Test against related membrane proteins in S. pombe to ensure target specificity. The systematic nature of S. pombe gene nomenclature allows for identification of potential cross-reactive targets.

• Application-specific validation: For Western blotting, confirm single band of expected molecular weight; for immunoprecipitation, verify enrichment of target protein through mass spectrometry; for ChIP applications, include appropriate controls as used in similar S. pombe chromatin studies .

• Reproducibility verification: Test multiple antibody lots to ensure consistent performance, which is particularly important when studying subtle phenotypes in heterochromatin maintenance .

These validation steps are essential as antibody performance can vary significantly between applications, and rigorous validation ensures robust, reproducible results in the specific experimental context of S. pombe research.

How can SPAC6F6.04c antibodies be optimized for chromatin immunoprecipitation (ChIP) studies?

Optimizing SPAC6F6.04c antibodies for ChIP studies requires specific methodological considerations:

• Fixation optimization: For membrane proteins like SPAC6F6.04c, crosslinking conditions may need to be modified from standard protocols. Test different fixation times (5-20 minutes) and formaldehyde concentrations (0.5-3%) to optimize crosslinking without over-fixation.

• Sonication parameters: Membrane proteins often require adjusted sonication protocols. Optimize cycle number and intensity to efficiently fragment chromatin while preserving epitope integrity. In S. pombe studies, 10-12 cycles of 30 seconds on/30 seconds off at medium intensity is often a good starting point .

• Antibody concentration titration: Test different antibody amounts (1-10 μg per ChIP reaction) to determine the optimal signal-to-noise ratio. This is particularly important when studying heterochromatin regions which can have higher background signals .

• Inclusion of appropriate controls: Include IgG controls, input samples, and when possible, samples from strains with tagged or deleted SPAC6F6.04c. The approaches used in studies of heterochromatin maintenance in S. pombe provide useful methodological guidelines .

• Buffer optimization: Consider specialized extraction buffers that are effective for membrane proteins while maintaining nuclear integrity. This may require higher detergent concentrations than standard ChIP protocols.

Following these optimization steps should enhance ChIP efficiency and specificity when working with SPAC6F6.04c antibodies in S. pombe studies.

How can SPAC6F6.04c antibodies be utilized in heterochromatin maintenance studies?

SPAC6F6.04c antibodies can provide valuable insights into heterochromatin dynamics in S. pombe, particularly when integrated with established approaches in this field. Based on research methodologies in S. pombe heterochromatin studies, consider the following applications:

• Protein localization analysis: Combine SPAC6F6.04c antibodies with ChIP assays to determine if this membrane protein associates with specific chromatin regions, similar to how Mrc1 association with heterochromatin has been documented .

• Temporal dynamics studies: Use synchronized cell populations to examine potential cell cycle-dependent associations of SPAC6F6.04c with heterochromatin, following approaches similar to those used to demonstrate that Mrc1 accumulates at the H3K14ac/H3K9me2 transition phase .

• Protein complex identification: Employ immunoprecipitation with SPAC6F6.04c antibodies followed by mass spectrometry to identify potential interaction partners, particularly looking for associations with known heterochromatin regulators such as SHREC components or HP1 proteins like Swi6 and Chp2 .

• Functional domain analysis: If working with mutant strains, SPAC6F6.04c antibodies can help determine which protein domains are essential for localization or function, similar to how the HBS domain of Mrc1 was identified as critical for heterochromatin maintenance .

These approaches allow researchers to position SPAC6F6.04c within the broader context of heterochromatin regulation networks in S. pombe, potentially revealing new functional connections to established pathways.

What advantages do recombinant SPAC6F6.04c antibodies offer over traditional antibodies?

Recombinant antibodies for SPAC6F6.04c provide several significant advantages over traditional polyclonal or hybridoma-derived monoclonal antibodies:

• Enhanced reproducibility: Being produced from defined genetic sequences, recombinant antibodies eliminate batch-to-batch variability that plagues traditional antibodies. This is particularly valuable for longitudinal studies of S. pombe chromatin dynamics .

• Improved specificity: Recombinant technology allows for selection of antibody sequences with optimal specificity, reducing cross-reactivity issues common with polyclonal antibodies. This is crucial when studying potentially redundant membrane proteins in S. pombe .

• Experimental flexibility: The availability of different conjugates and tags (such as biotin conjugation via Avi-tag) enables diverse experimental applications without compromising antibody performance .

• Ethical considerations: Recombinant antibody production reduces reliance on animal immunization, aligning with the 3Rs principles (Replacement, Reduction, Refinement) in research .

• Sequence transparency: Unlike hybridoma-derived antibodies that can contain contaminating light or heavy chains, recombinant antibodies have defined sequences, enhancing experimental transparency and reproducibility .

These advantages make recombinant SPAC6F6.04c antibodies particularly valuable for demanding applications such as flow cytometry and ChIP studies where specificity and reproducibility are essential for reliable data interpretation.

How might SPAC6F6.04c function interact with known heterochromatin maintenance mechanisms?

While specific interactions between SPAC6F6.04c and heterochromatin machinery are not directly described in the provided search results, we can formulate testable hypotheses based on established S. pombe heterochromatin maintenance mechanisms:

• Potential membrane-nuclear interface function: As a membrane protein, SPAC6F6.04c might participate in tethering heterochromatin regions to nuclear periphery structures, similar to how telomeres are anchored in other organisms. This could be tested by combining SPAC6F6.04c antibody immunofluorescence with fluorescence in situ hybridization (FISH) for heterochromatic regions.

• Cell cycle-dependent regulation: If SPAC6F6.04c shows cell cycle-dependent expression or modification patterns, it may participate in the S-phase derepression and subsequent re-establishment of heterochromatin observed in S. pombe . This could be investigated using synchronized cultures and examining protein levels/modifications throughout the cell cycle.

• Potential histone modification interactions: SPAC6F6.04c might influence histone modifications like H3K14ac or H3K9me2 that are crucial for heterochromatin dynamics in S. pombe . ChIP-seq experiments using both histone modification antibodies and SPAC6F6.04c antibodies could reveal correlations.

• SHREC complex interactions: Testing for physical interactions between SPAC6F6.04c and SHREC components (Clr1, Clr2, Clr3, or Mit1) using co-immunoprecipitation could reveal whether this membrane protein participates in recruiting chromatin-modifying complexes .

These hypotheses provide a framework for investigating potential roles of SPAC6F6.04c in S. pombe heterochromatin biology using the available antibodies as key research tools.

What are the optimal storage and handling conditions for SPAC6F6.04c antibodies?

To maintain optimal activity of SPAC6F6.04c antibodies, follow these research-validated storage and handling guidelines:

• Storage temperature: Store antibody aliquots at -20°C for long-term stability. Avoid repeated freeze-thaw cycles by preparing single-use aliquots during initial thawing .

• Working dilution storage: For diluted working solutions, store at 4°C and use within 1-2 weeks. Add preservatives such as 0.02% sodium azide for solutions stored longer than one week.

• Centrifugation recommendation: Briefly centrifuge the antibody vial before opening to collect all liquid at the bottom, especially after shipment or storage where material may adhere to the cap or walls.

• Thawing protocol: Thaw frozen antibodies slowly on ice rather than at room temperature to preserve protein structure and binding capacity.

• Handling precautions: Use clean, low-protein binding tubes and pipette tips to prevent protein adsorption and contamination. Consider using siliconized tubes for very dilute antibody solutions.

• Antibody dilution buffer: For optimal stability, dilute in buffers containing protein carriers (0.1-1% BSA or gelatin) to prevent nonspecific adsorption to surfaces.

Following these practices will help maintain antibody performance across experiments and extend the useful life of SPAC6F6.04c antibody reagents.

How can researchers troubleshoot weak or absent signals with SPAC6F6.04c antibodies?

When encountering signal problems with SPAC6F6.04c antibodies, consider this systematic troubleshooting approach:

• Epitope accessibility: Membrane proteins like SPAC6F6.04c may require optimized extraction conditions. Test different detergents (Triton X-100, CHAPS, DDM) at various concentrations to improve protein solubilization while maintaining epitope integrity.

• Fixation optimization: If using for immunofluorescence or ChIP, excessive fixation can mask epitopes. Test reduced fixation times or alternative fixatives (e.g., methanol instead of formaldehyde).

• Antibody concentration: Titrate antibody concentrations more broadly than standard protocols suggest. For Western blots, try ranges from 1:500 to 1:5000; for immunoprecipitation, test 1-10 μg per reaction.

• Signal enhancement strategies: Consider signal amplification methods such as:

  • Biotin-streptavidin systems using biotinylated secondary antibodies

  • Tyramide signal amplification (TSA)

  • Poly-HRP conjugated secondary antibodies

• Expression level verification: Confirm SPAC6F6.04c expression levels in your specific S. pombe strain and growth conditions using RT-qPCR, as expression may vary with environmental conditions or genetic background.

• Positive control inclusion: Include a sample with known high expression or a tagged version of SPAC6F6.04c to verify antibody functionality.

This systematic approach should help identify and address specific causes of signal problems when working with SPAC6F6.04c antibodies.

What control samples are essential when using SPAC6F6.04c antibodies in S. pombe research?

Rigorous experimental design for SPAC6F6.04c antibody use requires appropriate controls:

• Genetic knockout/knockdown controls: When available, include S. pombe strains with SPAC6F6.04c deletion or repression to establish signal specificity. This approach has been successfully used in heterochromatin studies in S. pombe to validate antibody specificity .

• Overexpression controls: Strains overexpressing SPAC6F6.04c provide positive controls and help establish the dynamic range of detection methods.

• Tagged protein controls: If available, strains expressing tagged versions of SPAC6F6.04c (e.g., with HA, FLAG, or GFP tags) allow validation using well-characterized tag antibodies.

• Isotype controls: Include appropriate isotype control antibodies at the same concentration as SPAC6F6.04c antibodies to establish background signal levels.

• Cell cycle phase controls: For studies examining cell cycle-dependent phenomena, include synchronized cultures at different cell cycle stages, as heterochromatin dynamics in S. pombe vary throughout the cell cycle .

• Cross-reactivity controls: Test antibody reactivity against closely related proteins or in related yeast species like S. cerevisiae (if the protein has orthologs) to assess specificity.

Incorporating these controls enhances data reliability and facilitates accurate interpretation of results, particularly important when studying potentially novel functions of SPAC6F6.04c in heterochromatin regulation or other cellular processes.