SPAC22H10.06c Antibody

What is SPAC22H10.06c and why is it studied in research?

SPAC22H10.06c is a gene in Schizosaccharomyces pombe (fission yeast) with the UniProt number Q10300 and Entrez Gene ID 2541880 . The protein encoded by this gene is studied in basic research on yeast cellular processes. Antibodies against this protein are valuable tools for investigating protein expression, localization, and function in S. pombe models. The study of this protein contributes to our understanding of fundamental cellular processes in eukaryotes, as S. pombe is an established model organism with conserved pathways relevant to human cell biology .

What are the technical specifications of commercially available SPAC22H10.06c antibodies?

The SPAC22H10.06c antibody is available as a rabbit polyclonal antibody purified by antigen affinity chromatography. Technical specifications include:

These specifications are important for researchers to consider when designing experiments and validating antibody performance .

How should I design validation experiments for SPAC22H10.06c antibody in my research?

For proper validation of SPAC22H10.06c antibody, implement a multi-step approach:

• Positive control testing: Use the provided recombinant antigen (200μg) as a positive control to confirm antibody binding specificity .

• Negative control validation: Employ the pre-immune serum (1ml) as a negative control to establish baseline signal and identify potential non-specific binding .

• Knockout/knockdown validation: When possible, use SPAC22H10.06c deletion strains or RNAi-depleted samples to confirm signal specificity.

• Multiple detection methods: Validate findings using orthogonal approaches such as combining Western blot with immunofluorescence or mass spectrometry.

• Cross-reactivity assessment: Test the antibody against lysates from related yeast species to determine specificity for S. pombe SPAC22H10.06c.

Document all validation experiments thoroughly, including antibody dilutions, exposure times, and image acquisition settings to establish reproducible protocols.

What are the optimal conditions for Western blot applications with SPAC22H10.06c antibody?

For optimal Western blot results with SPAC22H10.06c antibody, follow this methodological approach:

• Sample preparation:

  • Extract total proteins from S. pombe using either glass bead lysis or enzymatic digestion

  • Include protease inhibitors to prevent degradation

  • Prepare 20-50μg of total protein per lane

• Separation conditions:

  • Use 10-12% SDS-PAGE gels for optimal resolution

  • Include molecular weight markers appropriate for the expected protein size

• Transfer parameters:

  • Transfer to PVDF membrane at 100V for 1 hour or 30V overnight

  • Confirm transfer efficiency with reversible protein stain

• Blocking conditions:

  • Block with 5% non-fat dry milk or 3% BSA in TBST for 1 hour at room temperature

• Primary antibody incubation:

  • Start with 1:1000 dilution in blocking buffer

  • Incubate overnight at 4°C with gentle agitation

• Secondary antibody:

  • Use anti-rabbit HRP-conjugated antibody at 1:5000-1:10000

  • Incubate for 1 hour at room temperature

• Detection:

  • Use ECL substrate appropriate for expected expression level

  • Begin with short exposures (30 seconds) and increase as needed

Always include the provided positive control (recombinant antigen) and negative control (pre-immune serum) in initial optimization experiments to establish signal specificity .

How can I optimize ELISA protocols for quantitative detection of SPAC22H10.06c protein?

For quantitative ELISA application with SPAC22H10.06c antibody:

• Plate preparation:

  • Coat high-binding 96-well plates with capture antibody (2-5μg/ml) in carbonate buffer (pH 9.6)

  • Incubate overnight at 4°C

  • Wash 3× with PBST

• Standard curve generation:

  • Use the provided recombinant antigen to create a 7-point standard curve

  • Prepare 2-fold serial dilutions starting from 1000ng/ml

  • Include blank wells containing no antigen

• Sample preparation:

  • Process S. pombe lysates through mild detergent extraction

  • Prepare multiple dilutions (1:10, 1:50, 1:100) to ensure readings within standard curve range

• Detection optimization:

  • Test multiple dilutions of detection antibody (1:500-1:5000)

  • Optimize incubation times (1-2 hours at room temperature or overnight at 4°C)

  • Select appropriate substrate based on required sensitivity

• Technical considerations:

  • Run all standards and samples in triplicate

  • Include inter-plate calibration controls for multi-plate experiments

  • Calculate coefficient of variation (<15% acceptable)

For absolute quantification, generate a standard curve with each experiment and use four-parameter logistic regression for data analysis. Document all optimization steps to establish a reproducible protocol for your specific research conditions .

What approaches should I consider for studying protein-protein interactions involving SPAC22H10.06c?

To investigate protein-protein interactions involving SPAC22H10.06c:

• Co-immunoprecipitation (Co-IP):

  • Use SPAC22H10.06c antibody coupled to protein A/G beads

  • Perform pull-downs under native conditions

  • Analyze precipitated complexes by mass spectrometry

  • Validate interactions by reciprocal Co-IP

• Proximity labeling approaches:

  • Generate fusion proteins with BioID or APEX2

  • Identify proximal proteins through biotinylation and streptavidin pull-down

  • Confirm interactions with SPAC22H10.06c antibody

• Yeast two-hybrid screening:

  • Use SPAC22H10.06c as bait against S. pombe cDNA library

  • Validate positive interactions using Co-IP with the antibody

• Fluorescence microscopy:

  • Use SPAC22H10.06c antibody for immunofluorescence

  • Perform co-localization studies with putative interacting proteins

  • Consider proximity ligation assay (PLA) for direct interaction evidence

• Cross-linking mass spectrometry:

  • Apply protein cross-linkers to stabilize transient interactions

  • Immunoprecipitate with SPAC22H10.06c antibody

  • Identify cross-linked peptides by specialized MS/MS

For each approach, include appropriate controls and validate key findings using orthogonal methods to establish confidence in the identified interactions.

How can I address non-specific binding issues with SPAC22H10.06c antibody?

When encountering non-specific binding with SPAC22H10.06c antibody, implement this systematic troubleshooting approach:

• Antibody titration:

  • Test serial dilutions from 1:500 to 1:5000

  • Identify optimal concentration that maximizes specific signal while minimizing background

• Blocking optimization:

  • Compare different blocking agents (milk, BSA, commercial blockers)

  • Test longer blocking times (2-3 hours)

  • Consider adding 0.1-0.5% Tween-20 to reduce hydrophobic interactions

• Stringency adjustment:

  • Increase salt concentration in wash buffer (up to 500mM NaCl)

  • Add 0.1% SDS to reduce non-specific interactions

  • Perform longer or additional washing steps

• Pre-adsorption protocol:

  • Incubate antibody with non-target lysate

  • Remove non-specific antibodies by centrifugation

  • Use pre-adsorbed antibody solution for detection

• Control experiments:

  • Use pre-immune serum at the same concentration to identify non-specific binding

  • Include antigen competition assay where antibody is pre-incubated with excess antigen

Document all optimization steps and parameters to establish a reproducible protocol specific to your experimental system .

What standards should I follow for quantitative analysis of SPAC22H10.06c expression data?

For rigorous quantitative analysis of SPAC22H10.06c expression:

• Normalization approach:

  • Use multiple housekeeping proteins (e.g., actin, GAPDH, tubulin)

  • Consider total protein normalization (Ponceau, SYPRO Ruby)

  • Calculate relative expression using densitometry software

• Statistical analysis:

  • Run minimum of three biological replicates

  • Perform appropriate statistical tests based on data distribution

  • Report effect sizes along with p-values

• Dynamic range assessment:

  • Determine linear range of detection for your system

  • Ensure measurements fall within this range

  • Use multiple exposure times to capture wide dynamic range

• Quantification standards:

  • Include calibration curve using recombinant protein

  • Report absolute quantities when possible

  • Document all image acquisition parameters

• Reporting guidelines:

  • Present raw uncropped blots in supplementary materials

  • Include all replicates in analysis

  • Report antibody validation data

This methodological approach ensures reproducible and statistically sound quantification of SPAC22H10.06c expression across experimental conditions.

How does SPAC22H10.06c compare to orthologous proteins in other model organisms?

When comparing SPAC22H10.06c to orthologous proteins:

• Sequence homology:

  • Perform multiple sequence alignment with orthologues from S. cerevisiae, C. albicans, and other fungi

  • Identify conserved domains and motifs

  • Calculate percent identity and similarity scores

• Structural analysis:

  • Compare predicted or resolved structures

  • Identify conserved structural elements

  • Assess conservation of key functional sites

• Functional conservation:

  • Compare phenotypes of knockout/knockdown models across species

  • Evaluate conservation of interaction partners

  • Assess complementation potential in cross-species experiments

• Antibody cross-reactivity:

  • Test SPAC22H10.06c antibody against lysates from related species

  • Determine epitope conservation through sequence analysis

  • Document species-specificity for research applications

What considerations are important when integrating SPAC22H10.06c antibody data with other -omics approaches?

For integrating antibody-based data with other -omics approaches:

• Correlation with transcriptomics:

  • Compare protein levels detected by SPAC22H10.06c antibody with mRNA expression

  • Assess post-transcriptional regulation by calculating protein/mRNA ratios

  • Account for temporal delays between transcription and translation

• Integration with proteomics:

  • Validate mass spectrometry-based quantification with antibody detection

  • Compare relative abundances across detection methods

  • Document methodology-specific biases

• Pathway analysis integration:

  • Map SPAC22H10.06c to known pathways and protein complexes

  • Cross-reference antibody-detected interactions with interactome data

  • Identify potential functional modules

• Data normalization considerations:

  • Implement appropriate normalization for cross-platform comparisons

  • Account for differences in dynamic range between methods

  • Apply batch correction when integrating datasets

• Metadata documentation:

  • Maintain detailed experimental protocols

  • Record all sample preparation details

  • Document computational analysis parameters

This integrated approach provides a comprehensive understanding of SPAC22H10.06c function within the broader cellular context.

What strategies can improve detection sensitivity for low-abundance SPAC22H10.06c protein?

For enhanced detection of low-abundance SPAC22H10.06c:

• Sample enrichment techniques:

  • Implement subcellular fractionation to concentrate target compartment

  • Use immunoprecipitation to enrich SPAC22H10.06c before analysis

  • Apply TCA precipitation to concentrate total protein

• Signal amplification methods:

  • Utilize tyramide signal amplification (TSA) for immunodetection

  • Consider polymer-based detection systems with multiple HRP molecules

  • Use ultra-sensitive ECL substrates (femtogram detection range)

• Instrumentation optimization:

  • Employ cooled CCD cameras for chemiluminescence detection

  • Use long exposure times with low background systems

  • Consider fluorescent secondary antibodies with scanning detection

• Protocol modifications:

  • Extend primary antibody incubation (up to 48 hours at 4°C)

  • Reduce washing stringency slightly to preserve specific binding

  • Optimize transfer efficiency for high molecular weight proteins

• Data acquisition:

  • Use cumulative signal collection over multiple timepoints

  • Apply deconvolution algorithms to enhance signal-to-noise ratio

  • Implement image stacking for weak signals

Document all sensitivity optimization steps to establish reproducible protocols tailored to your specific experimental system .

How should experimental controls be designed for multi-condition experiments using SPAC22H10.06c antibody?

For robust multi-condition experiments:

• Internal loading controls:

  • Include multiple housekeeping proteins spanning different abundance ranges

  • Consider spike-in controls with known concentrations

  • Implement total protein normalization methods

• Antibody performance controls:

  • Include identical positive control lanes on all blots/plates

  • Run inter-experimental calibration samples

  • Monitor batch-to-batch antibody consistency

• Treatment validation controls:

  • Include molecular readouts confirming treatment efficacy

  • Document phenotypic changes corresponding to treatments

  • Measure known responder proteins as positive controls

• Technical variation controls:

  • Prepare master mixes for common reagents

  • Randomize sample processing order

  • Include technical replicates within and across experiments

• Statistical design considerations:

  • Determine appropriate sample size through power analysis

  • Plan for factorial experimental design when appropriate

  • Include time-course measurements for dynamic processes

This comprehensive control strategy ensures reliable data interpretation across experimental conditions and facilitates meaningful comparative analysis.

How might CRISPR-based approaches enhance functional studies of SPAC22H10.06c?

CRISPR technology offers several advanced approaches for SPAC22H10.06c research:

• Precise genomic modification:

  • Generate clean knockouts without marker genes

  • Create point mutations to study specific protein domains

  • Introduce regulatory element modifications to alter expression

• Endogenous tagging strategies:

  • Add fluorescent tags for live-cell imaging

  • Implement proximity-dependent labeling tags (BioID/TurboID)

  • Create degron-tagged versions for inducible protein depletion

• Validation approaches:

  • Use CRISPR-generated knockout lines to validate antibody specificity

  • Create allelic series to correlate antibody signal with expression level

  • Generate domain deletions to map antibody epitopes

• Multiplexed functional studies:

  • Implement CRISPR screens to identify genetic interactions

  • Create double mutants to study redundancy

  • Generate conditional alleles for essential genes

• Methodological considerations:

  • Design guides specific to S. pombe genome

  • Optimize homology-directed repair templates

  • Validate edits by sequencing and protein detection using the antibody

This integrated CRISPR approach can significantly enhance understanding of SPAC22H10.06c function while providing valuable tools for antibody validation .

What considerations are important when interpreting contradictory results from different detection methods for SPAC22H10.06c?

When facing contradictory results across detection methods:

• Epitope accessibility analysis:

  • Consider whether protein conformation affects epitope exposure

  • Evaluate effects of sample preparation on epitope availability

  • Assess potential post-translational modifications masking epitopes

• Method-specific biases:

  • Document detection limits for each method

  • Assess linear range of quantification for each technique

  • Consider differential sensitivity to protein modifications

• Antibody characteristics:

  • Evaluate whether antibody recognizes native vs. denatured protein

  • Consider multiple antibodies targeting different epitopes

  • Assess cross-reactivity with related proteins

• Biological variables:

  • Consider cell-type specific expression patterns

  • Evaluate subcellular localization differences

  • Assess potential isoform-specific detection

• Resolution approach:

  • Implement orthogonal validation techniques

  • Design experiments that directly address contradictions

  • Consider advanced methods like proximity labeling or FRET

This systematic analysis of contradictory results often leads to new biological insights regarding protein behavior under different experimental conditions.