SPCC63.05 Antibody

What is SPCC63.05 protein and what function does it serve in fission yeast?

SPCC63.05 is a protein found in Schizosaccharomyces pombe (fission yeast) that has been studied in various cellular contexts. While the search results don't provide specific functional information about this particular protein, fission yeast proteins are often researched as models for understanding fundamental eukaryotic processes. To determine its function, researchers often employ techniques such as gene knockout studies, localization experiments, and protein-protein interaction analyses.

When working with SPCC63.05 antibodies, it's important to consider the specificity of your reagent for detecting this protein in different experimental setups. Given that antibody characterization is critical for research reproducibility, any experiments should include appropriate controls to validate the specificity of the antibody for the target protein .

What validation methods should be used to confirm the specificity of SPCC63.05 antibody?

Antibody validation is essential, as approximately 50% of commercial antibodies fail to meet basic standards for characterization, leading to financial losses of $0.4-1.8 billion per year in the United States alone . For validating SPCC63.05 antibody, you should employ multiple complementary approaches:

• Genetic strategies: Use SPCC63.05 knockout or knockdown cells as negative controls to verify specificity.

• Orthogonal strategies: Compare antibody-dependent results with antibody-independent methods (e.g., mass spectrometry).

• Multiple antibody strategy: Use different antibodies against the same target and compare results.

• Recombinant expression: Overexpress the target protein and confirm increased signal.

• Immunocapture MS strategy: Use mass spectrometry to identify proteins captured by the antibody .

A properly validated antibody should demonstrate that it: (i) binds to the target protein; (ii) binds to the target protein in complex mixtures; (iii) does not bind to other proteins; and (iv) performs as expected under the specific experimental conditions employed .

What are the optimal conditions for using SPCC63.05 antibody in Western blot applications?

For optimal Western blot results with SPCC63.05 antibody, follow these methodological guidelines:

Sample preparation:

• Extract proteins from S. pombe using a suitable lysis buffer (e.g., RIPA buffer containing 1 mM PMSF, protease inhibitor cocktail, phosphatase inhibitor, and 0.05% NP-40) .

• For membrane proteins, consider specialized extraction protocols to maintain protein integrity.

Recommended protocol:

• Separate proteins using SDS-PAGE

• Transfer to nitrocellulose membrane

• Block with an appropriate blocking solution (typically 5% non-fat milk or BSA)

• Dilute SPCC63.05 antibody according to manufacturer recommendations; based on similar antibodies, start with 0.04-0.4 μg/mL dilution

• Incubate with primary antibody overnight at 4°C

• Wash thoroughly with TBST (3-5 times, 5-10 minutes each)

• Incubate with appropriate secondary antibody (e.g., Donkey Anti-Goat IgG(H+L) HRP) if the primary is goat-derived

• Develop using an ECL chemiluminescence system

Controls to include:

• Positive control: Wild-type S. pombe lysate

• Negative control: SPCC63.05 knockout/knockdown S. pombe lysate

• Loading control: Anti-α-tubulin antibody or other housekeeping protein

How should SPCC63.05 antibody be used in immunofluorescence applications for subcellular localization studies?

For subcellular localization studies using SPCC63.05 antibody in immunofluorescence, follow this methodological approach:

Sample preparation:

• Fix S. pombe cells using methanol fixation protocol

• Permeabilize cells appropriately to allow antibody access to intracellular targets

Staining protocol:

• Block with suitable blocking solution (e.g., 1% BSA in PBS)

• Apply primary SPCC63.05 antibody at recommended dilution (typically 0.25-2 μg/mL for similar antibodies)

• Incubate at 4°C overnight or room temperature for 1-2 hours

• Wash extensively with PBS/PBST

• Apply fluorophore-conjugated secondary antibody

• Counterstain with DAPI for nuclear visualization

• Mount slides with anti-fade mounting medium

Controls and validation:

• Include known markers for organelles to determine co-localization (e.g., ER, Golgi, mitochondria markers)

• Use genetic knockouts as negative controls

• Consider complementary approaches such as fractionation followed by Western blotting

When interpreting results, remember that membrane proteins like those found in the Golgi or post-Golgi may require specialized fixation protocols to preserve their native localization . Signal specificity should be confirmed using knockout controls or competing peptides.

How can SPCC63.05 antibody be used to study protein-protein interactions in fission yeast?

For investigating protein-protein interactions involving SPCC63.05, consider these methodological approaches:

Co-immunoprecipitation (Co-IP):

• Prepare cell lysate in a gentle lysis buffer (e.g., 10 mM Tris HCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40)

• Clear lysate by centrifugation (13,000 rpm, 30 min)

• Pre-clear with protein A/G beads

• Incubate with SPCC63.05 antibody (5-10 μg per 1 mg of protein)

• Add protein A/G beads and incubate (4°C, overnight)

• Wash beads thoroughly (3-5 times)

• Elute protein complexes with SDS loading buffer

• Analyze by SDS-PAGE followed by Western blot or mass spectrometry

Alternative approaches:

• Pil1 co-tethering assay: This imaging-based method for examining protein-protein interactions utilizes Pil1, a subunit of the plasma-membrane-associated eisosome complex, which forms visually distinctive filamentary structures in fission yeast .

• Yeast two-hybrid (Y2H) assay: Can be used to identify direct binding partners .

Validation strategies:

• Perform reciprocal Co-IPs

• Include negative controls (unrelated antibodies, IgG)

• Confirm interactions using orthogonal approaches (Y2H, proximity ligation assay)

• Verify with recombinant proteins in vitro

What approaches can be used to study SPCC63.05 protein function in autophagy pathways?

If investigating SPCC63.05 in relation to autophagy pathways (a significant area of fission yeast research), consider these methodological approaches:

Autophagy detection methods:

• GFP-Atg8 processing assay: Monitor the cleavage of GFP-Atg8 as an indicator of autophagy activity .

• Fluorescence microscopy: Track formation and movement of autophagosomes using fluorescently tagged autophagy markers.

• Proteomic analysis: Use LC-MS/MS to monitor changes in protein levels during autophagy induction .

Experimental design:

• Compare wild-type cells with SPCC63.05 knockout/knockdown cells

• Induce autophagy through nitrogen starvation (EMM2-N medium)

• Monitor autophagy markers at different time points

• Analyze using biochemical and imaging methods

Example protocol for inducing autophagy in S. pombe:

• Grow cells exponentially at 26°C in EMM2

• Harvest by vacuum filtration using a nitrocellulose membrane

• Wash with EMM2-N (EMM2 without nitrogen source)

• Resuspend in EMM2-N at a concentration of 2x10^6 or 5x10^6 cells/ml

• Incubate for 24h at 26°C

Data analysis:

• Quantify GFP-Atg8 processing efficiency

• Measure autophagosome formation rates

• Analyze cell viability under autophagy-inducing conditions

How can non-specific binding be minimized when using SPCC63.05 antibody in complex samples?

Non-specific binding is a common challenge with antibodies. For SPCC63.05 antibody, consider these approaches:

Optimization strategies:

• Blocking optimization: Test different blocking agents (BSA, non-fat milk, normal serum) and concentrations (1-5%).

• Antibody dilution: Titrate antibody concentrations to find the optimal signal-to-noise ratio.

• Buffer conditions: Adjust salt concentration (150-500 mM NaCl) and detergent levels (0.05-0.3% Tween-20 or Triton X-100).

• Pre-adsorption: Pre-incubate antibody with proteins from knockout cells to remove cross-reactive antibodies.

• Cross-adsorption: Similar to commercially available antibodies , consider using a cross-adsorbed version if non-specific binding persists.

Experimental validation:

• Always include a knockout/knockdown negative control

• Use competitive peptide blocking to confirm specificity

• Consider using multiple antibodies against the same target

Example optimization table for Western blotting:

Note: Optimal conditions are hypothetical and should be determined empirically for each specific application.

How can SPCC63.05 antibody be validated in the context of post-translational modifications?

Post-translational modifications (PTMs) can affect antibody recognition. To validate SPCC63.05 antibody for detecting modified forms of the protein:

Validation approaches:

• Enzymatic treatments: Treat samples with phosphatases, glycosidases (e.g., EndoH) , or other modification-removing enzymes to determine if antibody recognition is affected.

• Site-directed mutagenesis: Mutate potential modification sites and assess antibody recognition.

• Mass spectrometry: Identify modifications present on the protein and correlate with antibody detection patterns.

• Modification-specific antibodies: Use antibodies specific for common modifications (phospho, ubiquitin, etc.) alongside SPCC63.05 antibody.

Example protocol for glycosylation analysis:

• Divide protein sample into two aliquots

• Treat one aliquot with EndoH to remove N-linked glycans

• Compare antibody recognition between treated and untreated samples

• Analyze by Western blot to detect mobility shifts

PTM-specific considerations:

• For phosphorylation studies, include phosphatase inhibitors in lysis buffers

• For ubiquitination analysis, include deubiquitinase inhibitors

• For protein stability studies, treat cells with cycloheximide to block new protein synthesis

What controls are essential when using SPCC63.05 antibody in proteomics studies?

For proteomics studies involving SPCC63.05 antibody, implement these critical controls:

Essential controls:

• Genetic knockout/knockdown: The gold standard negative control to confirm antibody specificity .

• Isotype control: Use non-specific IgG matching the species and isotype of the SPCC63.05 antibody .

• Peptide competition: Pre-incubate antibody with immunizing peptide to block specific binding.

• Orthogonal detection method: Confirm findings using an antibody-independent method (e.g., targeted mass spectrometry).

• Technical replicates: Include at least three technical replicates for statistical validation.

• Independent antibody: Verify key findings with a different antibody targeting the same protein.

Experimental design recommendations:

• Include both biological and technical replicates

• Use appropriate statistical methods to analyze proteomics data

• Consider batch effects and normalize appropriately

Example validation workflow:

How can SPCC63.05 antibody be incorporated into studies of stress responses in fission yeast?

For studying stress responses in fission yeast using SPCC63.05 antibody, consider this methodological framework:

Experimental design:

• Stress induction: Apply relevant stressors (oxidative, temperature, nutritional) to S. pombe cultures.

• Time-course analysis: Collect samples at multiple time points after stress induction.

• Protein localization: Use immunofluorescence to track SPCC63.05 localization changes.

• Protein interactions: Perform Co-IP under normal vs. stressed conditions.

• Protein modifications: Analyze PTMs using appropriate techniques.

Example protocol for oxidative stress:

• Treat cells with H2DCFDA to detect reactive oxygen species accumulation

• Compare wild-type and mutant responses

• Track SPCC63.05 protein levels and localization changes

• Analyze using microscopy and biochemical approaches

Data integration:

• Correlate protein changes with transcriptome alterations

• Consider changes in protein interactions under stress

• Analyze modification patterns in response to different stressors

Functional validation:

• Test stress resistance in SPCC63.05 mutant strains

• Assess cell viability and morphology changes

• Measure relevant cellular processes (e.g., autophagy, cell wall integrity)

By following these guidelines and implementing appropriate controls, researchers can generate reliable and reproducible data using SPCC63.05 antibody in their experimental systems, contributing to our understanding of fission yeast biology and fundamental cellular processes.