SPCC757.13 Antibody

What is SPCC757.13 antibody and what organism does it target?

SPCC757.13 antibody (Product Code: CSB-PA524909XA01SXV) is a polyclonal antibody raised in rabbits against the recombinant Schizosaccharomyces pombe (strain 972 / ATCC 24843) SPCC757.13 protein. This antibody specifically targets the SPCC757.13 protein in fission yeast (S. pombe) . The antibody was developed using an antigen affinity purification method to ensure high specificity against the target protein, which has a UniProt accession number of O74923 .

What are the validated applications for SPCC757.13 antibody?

SPCC757.13 antibody has been validated for use in Western Blotting (WB) and Enzyme-Linked Immunosorbent Assay (ELISA) applications . When using this antibody for Western blotting, researchers should follow standard protocols for protein extraction from S. pombe, SDS-PAGE separation, and immunoblotting. Though not explicitly stated in the product documentation, antibodies of similar nature may potentially be used in immunoprecipitation or immunofluorescence studies following appropriate validation procedures.

What are the recommended storage conditions for SPCC757.13 antibody?

For optimal stability and activity retention, SPCC757.13 antibody should be stored at either -20°C or -80°C upon receipt . It is crucial to avoid repeated freeze-thaw cycles as these can lead to protein denaturation and loss of antibody activity. For laboratories that frequently use this antibody, it is recommended to prepare small aliquots for single use to minimize freeze-thaw cycles. The antibody is supplied in a storage buffer containing 0.03% Proclin 300 as a preservative and 50% glycerol in 0.01M PBS at pH 7.4 .

How should I optimize SPCC757.13 antibody concentration for Western blotting experiments?

When optimizing SPCC757.13 antibody for Western blotting experiments, researchers should perform a titration experiment to determine the optimal antibody concentration. While specific concentration recommendations for this antibody are not provided in the documentation, a general approach would be:

• Prepare a dilution series (e.g., 1:500, 1:1000, 1:2000, 1:5000) of the antibody

• Run identical protein samples on multiple Western blots

• Process each blot with a different antibody dilution

• Compare signal-to-noise ratios to determine optimal concentration

Based on similar antibodies, such as the Rabbit anti Dog Interleukin-13 antibody which is used at 0.1-2.0 μg/ml for Western blotting , researchers might start with a concentration range of 0.1-1.0 μg/ml for SPCC757.13 antibody.

What protein extraction methods are recommended for S. pombe when using SPCC757.13 antibody?

For optimal detection of the SPCC757.13 protein from S. pombe using this antibody, consider these extraction methods:

• Mechanical disruption: Using glass beads in a cell disruptor or bead beater in appropriate lysis buffer

• Enzymatic digestion: Pre-treatment with zymolyase to digest the cell wall, followed by detergent-based lysis

• Cryogenic grinding: Freezing cells in liquid nitrogen followed by grinding with mortar and pestle

The lysis buffer should contain protease inhibitors to prevent protein degradation and should be compatible with downstream applications. A typical buffer might include 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and a protease inhibitor cocktail.

How can I validate the specificity of SPCC757.13 antibody in my experiments?

To validate the specificity of SPCC757.13 antibody in your experiments, consider implementing these approaches:

• Positive and negative controls:

  • Positive control: Lysate from wild-type S. pombe expressing SPCC757.13

  • Negative control: Lysate from SPCC757.13 knockout strain (if available)

• Peptide competition assay: Pre-incubate the antibody with excess purified SPCC757.13 protein or peptide before application to your samples. Specific binding should be blocked.

• Multiple detection methods: Confirm results using different techniques (e.g., if using Western blot, also try immunofluorescence).

• Size verification: The detected band should correspond to the predicted molecular weight of SPCC757.13 protein.

What are common issues with Western blotting using SPCC757.13 antibody and how can they be resolved?

When performing Western blots with SPCC757.13 antibody, researchers might encounter various issues:

When troubleshooting, make incremental changes to your protocol and document all modifications systematically.

How can I differentiate between specific and non-specific binding when using SPCC757.13 antibody?

Differentiating between specific and non-specific binding is crucial for accurate data interpretation:

• Molecular weight verification: SPCC757.13-specific binding should appear at the predicted molecular weight of the protein.

• Knockout/knockdown validation: Compare results with samples where SPCC757.13 has been deleted or suppressed. Specific signals should be absent or significantly reduced.

• Peptide competition: Pre-incubating the antibody with purified SPCC757.13 protein should eliminate specific signals while non-specific signals may remain.

• Cross-species reactivity assessment: Test the antibody against lysates from other species. Since SPCC757.13 antibody is specific to S. pombe, it should not recognize proteins from evolutionarily distant organisms unless they share highly conserved epitopes.

How can SPCC757.13 antibody be used to study protein-protein interactions in S. pombe?

SPCC757.13 antibody can be leveraged for studying protein-protein interactions through several advanced techniques:

• Co-immunoprecipitation (Co-IP): Use the antibody to pull down SPCC757.13 protein along with its interacting partners, which can then be identified by mass spectrometry or Western blotting.

• Proximity Ligation Assay (PLA): Combine SPCC757.13 antibody with antibodies against putative interacting proteins to visualize interactions in situ with single-molecule resolution.

• Chromatin Immunoprecipitation (ChIP): If SPCC757.13 interacts with DNA or chromatin-associated proteins, ChIP using this antibody can identify genomic binding sites.

• Bimolecular Fluorescence Complementation (BiFC): While not directly using the antibody, this technique can complement antibody-based interaction studies by visualizing interactions in living cells.

This approach shares methodological similarities with studies of other proteins like SP17, which has been investigated for protein interactions in cancer research contexts .

Can SPCC757.13 antibody be used for immunofluorescence microscopy to study protein localization?

While ELISA and Western blotting are the validated applications for SPCC757.13 antibody , researchers interested in using it for immunofluorescence microscopy should:

• Perform validation experiments: Test different fixation methods (paraformaldehyde, methanol, acetone) and permeabilization techniques to determine optimal conditions.

• Optimize antibody concentration: Usually starting with a higher concentration than used for Western blotting, then titrating down.

• Include appropriate controls:

  • Primary antibody only (no secondary)

  • Secondary antibody only (no primary)

  • Pre-immune serum control

  • Peptide competition control

• Consider immunofluorescence enhancement techniques:

  • Tyramide signal amplification

  • Quantum dot conjugated secondary antibodies

  • Confocal microscopy with spectral unmixing

Similar to how IL-13 antibodies have been optimized for flow cytometry , researchers should conduct careful validation experiments before using SPCC757.13 antibody for new applications.

How should I adapt standard ELISA protocols for use with SPCC757.13 antibody?

When adapting standard ELISA protocols for SPCC757.13 antibody, consider the following modifications:

• Coating concentration optimization: Titrate the coating antigen (recombinant SPCC757.13 protein) concentration, typically starting at 1-5 μg/ml.

• Antibody dilution series: Create a dilution series of SPCC757.13 antibody (e.g., 1:500, 1:1000, 1:2000, 1:5000) to determine optimal concentration.

• Buffer optimization:

  • Coating buffer: 50 mM carbonate-bicarbonate buffer (pH 9.6)

  • Blocking buffer: 3-5% BSA or non-fat dry milk in PBS

  • Washing buffer: PBS with 0.05% Tween-20 (PBST)

  • Antibody diluent: 1% BSA in PBST

• Detection system selection: Choose appropriate HRP-conjugated secondary antibody against rabbit IgG, followed by colorimetric (TMB) or chemiluminescent substrate.

• Standard curve generation: If quantifying SPCC757.13 protein, prepare a standard curve using purified recombinant protein.

What considerations are important when using SPCC757.13 antibody in multiplexed assays?

When incorporating SPCC757.13 antibody into multiplexed assays where multiple targets are detected simultaneously, researchers should consider:

• Cross-reactivity assessment: Verify that SPCC757.13 antibody does not cross-react with other proteins in your experimental system or with other antibodies in the multiplex panel.

• Antibody isotype and host species compatibility: Ensure secondary detection reagents can distinguish between different primary antibodies in the panel. Using antibodies raised in different host species can facilitate this.

• Signal separation strategies:

  • If using fluorescent detection, select fluorophores with minimal spectral overlap

  • If using chromogenic detection, choose enzyme-substrate combinations with distinguishable colors

  • Consider sequential detection protocols if cross-reactivity is a concern

• Validation of multiplex results: Confirm findings from multiplexed assays with single-plex experiments to ensure accuracy.

• Data normalization: Develop appropriate controls and normalization strategies to account for variations in antibody efficiency and target abundance.

This approach draws on methodology principles similar to those used in complex immunological studies, such as the evaluation of IL-13 in immunoregulatory contexts .