SPCC622.02 Antibody

What is SPCC622.02 Antibody and what is its target?

SPCC622.02 Antibody (catalog code CSB-PA527235XA01SXV) is a research antibody that targets a putative uncharacterized membrane protein in Schizosaccharomyces pombe (strain 972/ATCC 24843), commonly known as fission yeast . The target protein is identified in the UniProt database with accession number O94592 and is designated by the systematic ID SPCC622.02 in the S. pombe genome . This antibody serves as an important research tool for studying membrane protein biology in this model organism.

What validation methods should be applied to SPCC622.02 Antibody before use?

At minimum, SPCC622.02 Antibody should undergo application-specific validation following the consensus "5 pillars" approach developed for antibody validation . These include:

• Genetic validation: Using CRISPR/knockout strains of S. pombe lacking SPCC622.02 to confirm antibody specificity

• Orthogonal validation: Correlating protein levels detected by the antibody with mRNA expression data

• Independent antibody validation: Using multiple antibodies targeting different epitopes of the same protein

• Expression validation: Testing the antibody against recombinant SPCC622.02 protein

• Immunocapture-mass spectrometry: Confirming target specificity through peptide sequencing

A combination of these approaches provides greater confidence in antibody specificity. Researchers should clearly document which validation methods were performed and include detailed methodology in publications .

What are common applications for SPCC622.02 Antibody in fission yeast research?

Based on antibody applications for similar membrane proteins in S. pombe, SPCC622.02 Antibody can be utilized in:

• Western blotting: For detecting and quantifying protein expression levels

• Immunoprecipitation: For studying protein-protein interactions

• Immunofluorescence: For visualizing subcellular localization

• ChIP assays: If the protein has DNA-binding properties

• Flow cytometry: For quantitative analysis in cell populations

Each application requires specific optimization and validation, as antibody performance can vary significantly between applications due to differences in how the epitope is presented . Application-specific validation is essential, as the conformation of the target antigen changes between methods (e.g., denatured in western blotting versus native in immunoprecipitation) .

What factors affect SPCC622.02 Antibody specificity in different experimental contexts?

Several factors can influence antibody specificity when working with SPCC622.02:

Researchers should be particularly attentive to the possibility of off-target binding, as many antibodies recognize additional molecules beyond their intended target . This is especially important for putative uncharacterized proteins like SPCC622.02, where complete structural information may be lacking .

How can researchers address batch-to-batch variability with SPCC622.02 Antibody?

Batch-to-batch variability is a significant challenge with biological reagents like antibodies . To address this with SPCC622.02 Antibody:

• Record lot numbers: Always document the specific antibody lot used in experiments

• Validate each new lot: Re-validate every new batch against a previously characterized batch

• Create internal reference standards: Prepare and freeze lysates from wild-type S. pombe as positive controls

• Generate validation datasets: Document antibody performance metrics for each batch

• Consider long-term planning: Purchase larger quantities of a validated lot for long-term studies

The research community would benefit from sharing validation data for specific lots of SPCC622.02 Antibody, as this improves reproducibility across laboratories . Maintaining detailed records of antibody performance characteristics helps identify whether experimental variability stems from biological differences or reagent inconsistencies.

What are optimal immunoprecipitation conditions for SPCC622.02 Antibody?

While specific optimization is necessary for each research context, general guidelines for immunoprecipitation with SPCC622.02 Antibody in S. pombe include:

• Lysis buffer selection: Use buffers optimized for membrane proteins, containing appropriate detergents (e.g., NP-40, Triton X-100, or more specialized detergents for membrane proteins)

• Antibody concentration: Typically 2-5 μg per 500 μg-1 mg of total protein

• Incubation conditions: Overnight at 4°C with gentle rotation

• Washing stringency: Balance between removing non-specific binding while preserving specific interactions

• Controls: Include no-antibody controls and, ideally, samples from SPCC622.02 knockout strains

For subsequent mass spectrometry analysis, consider that the sequenced peptides will include both directly captured antigens and interacting proteins . The top three peptide sequences should all come from the target protein to provide good evidence of antibody selectivity .

How should experiments be designed to compare wild-type and mutant SPCC622.02 protein expression?

Robust experimental design for comparing wild-type and mutant SPCC622.02 protein expression should include:

• Multiple biological replicates: Minimum of three independent cultures

• Technical replicates: At least duplicate analyses of each biological sample

• Appropriate controls:

  • Positive control: Wild-type S. pombe lysate

  • Negative control: SPCC622.02 knockout strain

  • Loading control: Constitutively expressed protein unaffected by experimental conditions

• Quantification method: Use digital imaging and software analysis rather than visual assessment

• Statistical analysis: Apply appropriate statistical tests with correction for multiple comparisons

• Blinded analysis: When possible, blind the researcher performing quantification to sample identity

Document all experimental parameters, including growth conditions, cell density at harvest, lysis method, protein quantification method, gel percentage, transfer conditions, antibody dilutions, and exposure times . This comprehensive documentation facilitates reproducibility and allows meaningful comparison between experiments.

What considerations are important when using SPCC622.02 Antibody for co-localization studies?

For co-localization studies using SPCC622.02 Antibody in immunofluorescence microscopy:

• Fixation optimization: Test multiple fixation methods (e.g., paraformaldehyde, methanol) to preserve both membrane structure and epitope accessibility

• Permeabilization: Optimize detergent type and concentration for membrane proteins

• Multiplexing considerations:

  • Select secondary antibodies with minimal spectral overlap

  • When using multiple primary antibodies, ensure they are raised in different species

  • Consider sequential staining if antibodies have potential cross-reactivity

• Controls for specificity:

  • Single-color controls to assess bleed-through

  • Secondary-only controls to assess non-specific binding

  • SPCC622.02 knockout strains as negative controls

• Image acquisition settings:

  • Maintain consistent settings between samples

  • Capture images below saturation

  • Use appropriate resolution for co-localization analysis

• Quantitative analysis:

  • Apply statistical measures of co-localization (e.g., Pearson's coefficient)

  • Report both visual and quantitative assessments

  • Include spatial distribution analysis where relevant

These considerations help ensure that observed co-localization reflects true biological associations rather than technical artifacts or non-specific binding .

What are common causes of inconsistent results with SPCC622.02 Antibody?

Inconsistent results when using SPCC622.02 Antibody may stem from several sources:

When troubleshooting, change only one variable at a time and maintain detailed records of all modifications to protocols . This systematic approach helps identify the specific factors contributing to inconsistent results.

How can contradictory findings with SPCC622.02 Antibody across different labs be reconciled?

Reconciling contradictory findings requires a systematic approach:

• Compare antibody details: Verify that the same antibody (including company, catalog number, and lot) was used

• Review validation methods: Assess how each laboratory validated the antibody for their specific application

• Examine experimental conditions:

  • Cell growth and harvesting conditions

  • Sample preparation methods

  • Protocol details (concentrations, incubation times, temperatures)

  • Detection systems and sensitivity

• Consider biological variables:

  • Strain differences (even within S. pombe 972)

  • Growth phase effects

  • Media composition impacts

  • Environmental stressors

• Collaborative cross-validation:

  • Exchange protocols and reagents

  • Perform identical experiments in different laboratories

  • Share raw data for independent analysis

The research community's openness about antibody performance characteristics and validation methods is crucial for improving reproducibility . When publishing, include detailed methods and antibody validation data to facilitate cross-laboratory comparisons.

What approaches can optimize signal-to-noise ratio in experiments using SPCC622.02 Antibody?

To optimize signal-to-noise ratio:

• Antibody titration: Test a range of concentrations to identify the minimum concentration that provides specific signal

• Blocking optimization:

  • Test different blocking agents (BSA, milk, serum, commercial blockers)

  • Optimize blocking time and temperature

  • Consider adding detergents to blocking buffer

• Sample preparation refinement:

  • Optimize lysis conditions for membrane proteins

  • Consider enrichment methods for membrane fractions

  • Remove interfering substances through additional purification steps

• Washing optimization:

  • Increase number of washes

  • Modify wash buffer composition

  • Extend wash durations

• Detection system selection:

  • Choose detection methods appropriate to expected expression level

  • Consider signal amplification methods for low-abundance targets

  • Use quantitative detection systems with broad dynamic range

• Advanced approaches:

  • Pre-adsorption of antibody with non-specific proteins

  • Affinity purification of antibody

  • Subtraction of signal from knockout controls

Maintaining optimal signal-to-noise ratio is particularly important for quantitative analyses and when studying proteins with low expression levels or in complex sample matrices .

What information should be included in Methods sections when reporting experiments using SPCC622.02 Antibody?

Complete Methods sections should include:

• Antibody details:

  • Supplier and catalog number

  • Lot number

  • Clone number (if monoclonal)

  • Host species and antibody type

  • Concentration used

• Validation performed:

  • Which of the "5 pillars" were implemented

  • Positive and negative controls used

  • Previous publications validating this antibody (if applicable)

• Experimental conditions:

  • Complete protocol details with concentrations and times

  • Sample preparation specifics

  • Equipment and settings used

  • Image acquisition parameters

• Data analysis:

  • Quantification methods

  • Software used (with version)

  • Statistical approaches

  • Blinding procedures (if applicable)

This comprehensive reporting enables other researchers to accurately replicate experiments and properly interpret results . Consider including key validation data in supplementary materials if space in the main text is limited.

How should researchers address discrepancies between antibody-based and genetic approaches in studying SPCC622.02?

When antibody-based approaches (e.g., immunoblotting, immunofluorescence) yield different results from genetic approaches (e.g., fluorescent protein tagging, transcriptomics):

This comprehensive and transparent approach advances scientific understanding even when results appear contradictory . Discrepancies often lead to new insights about protein biology, technical limitations, or novel regulatory mechanisms.