SPCC569.06 Antibody

What is SPCC569.06 and what organism does it originate from?

SPCC569.06 is a gene/protein identifier from Schizosaccharomyces pombe (fission yeast). This nomenclature follows the standard S. pombe genome annotation format "SP[chromosome][contig].[gene number]", where "SP" indicates S. pombe, "CC" denotes chromosome location, "569" represents the specific contig, and "06" indicates the gene number within that contig. The protein encoded by this gene is the target of the SPCC569.06 antibody, which has been developed specifically for detecting this fission yeast protein in experimental applications.

What applications has the SPCC569.06 antibody been validated for?

The SPCC569.06 antibody has been specifically validated for Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blotting (WB) applications . These validation studies confirm the antibody's ability to specifically recognize the target protein in both native and denatured states, making it suitable for quantitative protein detection (ELISA) and size-based protein identification (Western blot). Researchers should note that while additional applications may be possible, they would require independent validation.

What is the proper storage protocol for SPCC569.06 antibody?

The SPCC569.06 antibody should be stored at -20°C to -80°C upon receipt to maintain optimal activity . After reconstitution, the antibody can be stored at -20°C to -80°C for up to 6 months under sterile conditions. Researchers should avoid repeated freeze-thaw cycles, as these can diminish antibody performance through protein denaturation and aggregation. For short-term storage (up to one month), the reconstituted antibody may be kept at 2-8°C under sterile conditions .

What are the host species and isotype characteristics of this antibody?

The SPCC569.06 antibody is a rabbit polyclonal antibody of the IgG isotype . The polyclonal nature means it contains a heterogeneous mixture of antibodies that recognize multiple epitopes on the target protein, potentially increasing detection sensitivity but with some variability between lots. The rabbit host species provides advantages including robust immune responses to diverse antigens and compatibility with many secondary detection systems in common laboratory applications.

What is the optimal dilution range for Western blot applications?

While specific dilution recommendations can vary between lots, polyclonal antibodies like SPCC569.06 typically perform optimally in Western blotting at dilutions ranging from 1:500 to 1:2000. Researchers should perform a dilution series (e.g., 1:500, 1:1000, 1:2000, 1:5000) to empirically determine the optimal concentration that maximizes specific signal while minimizing background for their particular experimental conditions, sample type, and detection system.

What positive controls should be included when working with this antibody?

The SPCC569.06 antibody is supplied with 200 μg of recombinant immunogen protein/peptide that serves as an ideal positive control . This control should be included in initial validation experiments to confirm antibody performance. Additionally, researchers should consider using wild-type S. pombe cell lysates as biological positive controls. For negative controls, lysates from SPCC569.06 deletion strains (if available) or non-S. pombe species lysates can help confirm specificity.

How should protein extraction be optimized for SPCC569.06 detection?

For optimal detection of SPCC569.06 protein from S. pombe, researchers should consider the following extraction protocol:

Table 1: Recommended Protein Extraction Protocol for SPCC569.06 Detection

This protocol maximizes the extraction of intact SPCC569.06 protein while minimizing proteolytic degradation that could affect antibody recognition.

Can the SPCC569.06 antibody be adapted for immunoprecipitation studies?

While not explicitly validated for immunoprecipitation (IP), many rabbit polyclonal IgG antibodies can perform effectively in IP applications. Researchers attempting to use SPCC569.06 antibody for IP should consider the following optimization strategy:

• Start with higher antibody concentrations (5-10 μg per 500 μg of protein lysate)

• Use gentle lysis conditions to preserve protein interactions

• Pre-clear lysates with Protein A/G beads to reduce non-specific binding

• Extend antibody-antigen binding incubation to overnight at 4°C

• Validate IP results with Western blotting using the same antibody

Success in IP depends on epitope accessibility in the native protein conformation, which cannot be predicted without empirical testing.

How can specificity be verified in different experimental contexts?

Verifying SPCC569.06 antibody specificity is critical for experimental validity. Multiple approaches should be employed:

Table 2: Strategies for Verifying SPCC569.06 Antibody Specificity

What are the considerations for using this antibody in chromatin immunoprecipitation (ChIP) studies?

While the SPCC569.06 antibody has not been explicitly validated for ChIP applications, researchers investigating transcription-related functions may consider adapting it for this purpose. Successful adaptation would require:

• Confirmation that the antibody recognizes the native protein conformation

• Optimization of crosslinking conditions (typically 1% formaldehyde for 10-15 minutes)

• Sonication parameters adjusted for S. pombe chromatin (typically 10-15 cycles)

• Higher antibody concentrations than used for Western blotting (10-15 μg per experiment)

• Rigorous controls including no-antibody and IgG-only immunoprecipitations

The success of ChIP applications cannot be guaranteed without empirical validation, as epitope accessibility can be severely affected by formaldehyde crosslinking.

What are the most common causes of weak or absent signals when using this antibody?

When encountering signal problems with SPCC569.06 antibody, researchers should systematically evaluate:

Table 3: Troubleshooting Guide for SPCC569.06 Antibody

This systematic approach allows researchers to identify and address the specific factors affecting antibody performance in their experimental system.

How does buffer composition affect antibody performance?

Buffer composition can significantly impact SPCC569.06 antibody performance. The antibody is provided in a buffer containing 0.03% Proclin 300 and 50% Glycerol , which helps maintain stability during storage. For experimental applications, consider the following buffer effects:

• Blocking agents: BSA is generally preferred at 3-5% for Western blotting; milk proteins may contain phosphatases that could affect phospho-epitope detection

• Detergents: Low concentrations (0.05-0.1%) of Tween-20 reduce non-specific binding in wash buffers

• Salt concentration: 150-500 mM NaCl helps reduce non-specific ionic interactions

• pH sensitivity: Most antibody-antigen interactions perform optimally at neutral pH (7.0-7.5)

• Reducing agents: DTT or β-mercaptoethanol can disrupt antibody disulfide bonds if present during incubation

Optimizing these buffer components can dramatically improve signal-to-noise ratio in antibody-based detection systems.

What is the expected molecular weight for SPCC569.06 protein in Western blots?

While the exact molecular weight of SPCC569.06 protein is not explicitly stated in the provided information, researchers can calculate the theoretical molecular weight from the protein sequence. For fission yeast proteins, differences between observed and predicted molecular weights may occur due to:

• Post-translational modifications (phosphorylation, glycosylation, etc.)

• The presence of highly charged amino acid clusters affecting SDS binding

• Protein structural elements that persist despite denaturation

When first using the antibody, researchers should run appropriate molecular weight markers and anticipate potential size variations from theoretical predictions based on these factors.

How should researchers interpret multiple bands in Western blots?

Multiple bands in Western blots using SPCC569.06 antibody may represent:

• Isoforms: Alternative splice variants of the target protein

• Post-translational modifications: Phosphorylation, glycosylation, or other modifications

• Proteolytic fragments: Degradation products from sample processing

• Cross-reactivity: Binding to proteins with similar epitopes

To distinguish between these possibilities, researchers should conduct validation experiments including:

• Treatment with phosphatases to remove phosphorylation

• Deglycosylation enzymes to remove glycosylation

• Expression of recombinant forms of specific isoforms

• Comparative analysis in deletion/knockout strains

These approaches help establish which bands represent specific detection of the target protein versus artifacts or cross-reactivity.

What methods can enhance quantitative analysis when using this antibody?

For quantitative applications of SPCC569.06 antibody, researchers should implement:

Table 4: Strategies for Quantitative Analysis Using SPCC569.06 Antibody

These approaches transform qualitative antibody detection into robust quantitative data suitable for comparative analysis across experimental conditions.