SPBP16F5.05c Antibody

What is SPBP16F5.05c and what cellular functions does it regulate?

SPBP16F5.05c (Pof1) is an essential F-box protein in Schizosaccharomyces pombe that forms part of the SCFPof1 ubiquitin ligase complex. It contains an F-box motif and WD40 repeat domains, suggesting its role in substrate recognition and protein-protein interactions. Functionally, Pof1 regulates the stability of the transcription factor Zip1, which is involved in cadmium response pathways . Temperature-sensitive mutations of Pof1 result in cell cycle arrest with characteristic small cell morphology, indicating its importance in cell cycle regulation.

When studying this protein, researchers should be aware that Pof1 mutants (particularly pof1-6 and pof1-12) show accumulation of slower migrating forms of Zip1, suggesting that Pof1 regulates post-translational modifications of its substrates in addition to protein turnover .

What are the most common epitopes targeted when generating antibodies against SPBP16F5.05c?

For generating antibodies against SPBP16F5.05c, researchers typically target:

• The F-box domain (amino acids 100-150) - functionally significant but may be occluded when bound to Skp1

• The WD40 repeat region - involved in substrate binding

• The N-terminal or C-terminal regions - often more accessible in the native protein

When planning epitope selection, consider that mutations in the F-box motif (F109S and S118P) and in regions flanking the WD40 repeats (K246E and S566G) have been shown to disrupt Pof1 function , making these regions potentially important for antibody recognition of functionally relevant conformations.

How do I validate the specificity of an anti-SPBP16F5.05c antibody?

A robust validation approach for anti-SPBP16F5.05c antibodies should include:

• Western blot analysis comparing wild-type cells with pof1 mutants or knockdown strains

• Immunoprecipitation followed by mass spectrometry to confirm target identity

• Immunofluorescence microscopy comparing localization patterns with GFP-tagged Pof1

• Cross-reactivity tests with related F-box proteins

Validation Data Example:

What are the optimal conditions for using anti-SPBP16F5.05c antibodies in immunoprecipitation experiments?

When performing immunoprecipitation of SPBP16F5.05c/Pof1, consider the following optimized protocol based on experimental findings:

• Cell lysis conditions: Use lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% NP-40, 10% glycerol, with protease and phosphatase inhibitors

• Antibody binding: Incubate lysates with antibody at 4°C for 2-4 hours

• Precipitation: Add protein A/G beads and incubate for an additional 1-2 hours

• Washing: Use stringent washing (high salt buffer followed by detergent buffer) to minimize non-specific binding

• Elution: Perform sequential elution with increasing stringency

For co-immunoprecipitation studies targeting SCFPof1 complex components, note that detergent choice is critical - stronger detergents may disrupt protein-protein interactions within the complex . When investigating Pof1-substrate interactions, consider using proteasome inhibitors (MG132) or temperature-sensitive mutations (as in the mts3-1 proteasome mutant strain) to stabilize otherwise transient interactions .

How can I use anti-SPBP16F5.05c antibodies to study protein degradation pathways?

To study SPBP16F5.05c/Pof1-mediated protein degradation:

• Cycloheximide chase assays: Antibodies can be used to monitor substrate turnover rates by western blotting. Research has shown that in pof1-6 mutants, Zip1 has an increased half-life compared to wild-type cells (see quantification in Figure 4B of reference) .

• Ubiquitination assays: Immunoprecipitate the substrate (e.g., Zip1) and probe for ubiquitin to detect polyubiquitination dependent on functional Pof1.

• Post-translational modification mapping: Use the antibody to purify the protein and analyze by mass spectrometry to identify regulatory modifications.

Experimental example: When studying Zip1 degradation dynamics in wild-type versus pof1-6 mutant cells, researchers observed that the half-life of the faster migrating Zip1-HA band increased significantly in the mutant (Figure 4A-B). Interestingly, slower migrating forms of Zip1-HA were still degraded efficiently in pof1-6, suggesting a complex regulation mechanism .

What controls should I include when performing immunofluorescence with anti-SPBP16F5.05c antibodies?

For rigorous immunofluorescence experiments with anti-SPBP16F5.05c antibodies, include:

• Genetic controls:

  • Wild-type cells (positive control)

  • pof1 mutant strains (negative control)

  • GFP-tagged Pof1 strain (co-localization control)

• Technical controls:

  • Secondary antibody only (background control)

  • Pre-immune serum (non-specific binding control)

  • Peptide competition assay (epitope specificity control)

• Biological controls:

  • Cell cycle synchronized samples (to detect cell-cycle dependent localization)

  • Stress conditions such as cadmium exposure (to observe potential relocalization)

For quantitative analysis, measure the nuclear/cytoplasmic ratio of staining intensity across multiple cells (n>100) in different conditions to detect subtle localization changes.

How can I determine if post-translational modifications affect antibody recognition of SPBP16F5.05c?

To assess the impact of post-translational modifications on antibody recognition:

• Phosphorylation analysis: Treat immunoprecipitated SPBP16F5.05c with λ-phosphatase (as demonstrated for Zip1 in Figure 4C ) and compare antibody reactivity before and after treatment.

• Site-directed mutagenesis: Generate point mutations at predicted modification sites and compare antibody binding.

• Mass spectrometry approach: Use a workflow combining:

  • Enrichment of modified forms

  • MS/MS analysis to identify modification sites

  • Correlation of modification status with antibody reactivity

Research findings: Studies with Zip1 showed that multiple bands in western blots corresponded to differentially phosphorylated forms, as treatment with λ-phosphatase collapsed the pattern to a single band . Similar approaches can be applied to SPBP16F5.05c to understand its modification landscape and how it affects antibody recognition.

What strategies can I use to improve the affinity of existing anti-SPBP16F5.05c antibodies?

For antibody affinity maturation against SPBP16F5.05c, consider these advanced approaches:

• Computational antibody design protocols:

  • Use IsAb protocol which integrates RosettaAntibody for structure prediction, RosettaRelax for energy minimization, and two-step docking

  • Apply alanine scanning to identify hotspots for targeted mutagenesis

  • Perform computational affinity maturation to optimize binding properties

• High-throughput single-cell sequencing approach:

  • Isolate B cells from immunized subjects

  • Perform scRNA-seq and VDJ sequencing to identify antigen-binding clonotypes

  • Express and characterize top candidate sequences

  • Test for nanomolar binding affinity using Biolayer Interferometry

• Machine learning and supercomputing:

  • Apply machine learning models trained on antibody-antigen interaction data

  • Use supercomputing resources to predict structural interactions

  • Design mutations predicted to optimize binding

These methods have demonstrated success in developing high-affinity antibodies with KD values in the nanomolar range (e.g., 1.959 × 10^-9 M for the Abs-9 antibody against SpA5 ).

How do I resolve contradictions between immunoprecipitation and immunofluorescence data for SPBP16F5.05c?

When facing discrepancies between IP and IF results:

• Epitope accessibility analysis:

  • Different fixation methods can mask or expose epitopes

  • Compare crosslinking agents (formaldehyde, DSP, glutaraldehyde)

  • Test epitope retrieval methods (heat, pH, detergents)

• Conformational considerations:

  • IP may capture denatured forms while IF detects native conformation

  • Use antibodies targeting different epitopes to compare results

  • Consider developing conformation-specific antibodies

• Complex formation interference:

  • In SCFPof1 complexes, the F-box domain interacts with Skp1 while WD40 repeats bind substrates

  • Determine if antibody epitopes overlap with protein interaction surfaces

  • Test cell fractionation to separate bound vs. unbound pools

Methodological solution: Validate results using orthogonal approaches, such as complementing antibody-based detection with GFP-tagged Pof1 visualization. Temperature-sensitive mutants (pof1-6, pof1-12) can be valuable tools to confirm specificity under various conditions .

How can I accurately quantify western blot data when analyzing SPBP16F5.05c levels across different experimental conditions?

For precise quantification of SPBP16F5.05c western blot data:

• Normalization strategy:

  • Use total protein normalization rather than single housekeeping proteins

  • Consider multiple loading controls (e.g., Cdc2 as used in Figure 4A )

  • Apply REVERT total protein stain for lane normalization

• Technical considerations:

  • Ensure linear dynamic range by performing dilution series

  • Use standardized exposure times and avoid saturated signals

  • Apply statistical methods to assess significance across biological replicates

• Software analysis:

  • Use densitometry software with background subtraction

  • Apply rolling ball algorithm for accurate band detection

  • Generate quantification graphs with error bars (as shown in Figure 4B )

Quantification example: When analyzing Zip1 stability in wild-type versus pof1-6 mutants, researchers quantified upper and lower bands separately, allowing them to discover differential degradation kinetics for the different forms of the protein .

What bioinformatic approaches can help predict epitopes for generating new anti-SPBP16F5.05c antibodies?

Advanced bioinformatic approaches for epitope prediction include:

• Structural prediction methods:

  • AlphaFold2-based modeling of SPBP16F5.05c structure

  • Molecular docking simulations to identify accessible regions

  • Epitope validation using competitive binding assays

• Sequence-based analysis:

  • Hydrophilicity plots and surface probability analysis

  • Conservation analysis across homologs to identify functionally important regions

  • Intrinsic disorder prediction to locate flexible, accessible regions

• Machine learning integration:

  • Combine features from sequence and structural predictions

  • Train models on known antibody-antigen complexes

  • Validate predictions using experimental antibody binding data

Research application: The Antigen-Antibody Complex Database (AACDB) contains 7,498 manually processed antigen-antibody complexes with comprehensive paratope and epitope annotations , providing valuable benchmarking data for epitope prediction algorithms.

How do I interpret cross-reactivity of anti-SPBP16F5.05c antibodies with related F-box proteins?

When analyzing potential cross-reactivity:

• Sequence similarity analysis:

  • Perform multiple sequence alignment of SPBP16F5.05c with other F-box proteins

  • Identify regions with high conservation that might cause cross-reactivity

  • Design epitope selection to avoid these regions when possible

• Structural homology consideration:

  • Compare predicted or known structures of related F-box proteins

  • Identify unique structural features of SPBP16F5.05c

  • Assess accessibility of distinguishing features

• Experimental validation approaches:

  • Test antibody against recombinant related F-box proteins

  • Perform immunodepletion experiments

  • Use genetic knockouts/knockdowns to confirm specificity

Interpretation framework:

What are common issues with anti-SPBP16F5.05c antibodies and how can they be resolved?

Common challenges with SPBP16F5.05c antibodies include:

• Weak signal in western blots:

  • Resolution: Optimize extraction buffers to include phosphatase inhibitors and denaturants

  • Research finding: Temperature-sensitive pof1 mutants show increased levels of both Pof1 and its substrate Zip1 , making these strains useful positive controls

• High background in immunofluorescence:

  • Resolution: Implement more stringent blocking with 5% BSA and 2% normal serum

  • Additional approach: Use peptide competition assays to determine specificity

• Inconsistent immunoprecipitation results:

  • Resolution: Pre-clear lysates and optimize salt/detergent concentrations

  • Experimental insight: When studying protein-protein interactions, consider using proteasome mutants (e.g., mts3-1) to stabilize otherwise transient complexes

• Poor reproducibility across experiments:

  • Resolution: Standardize cell growth conditions and lysis procedures

  • Important consideration: Cell cycle phase can significantly affect F-box protein expression and localization

• Antibody batch variation:

  • Resolution: Implement validation protocols for each new lot

  • Best practice: Maintain reference samples from successful experiments for side-by-side comparisons

How do experimental conditions affect the performance of anti-SPBP16F5.05c antibodies?

Various experimental conditions can significantly impact antibody performance:

• Temperature effects:

  • Temperature-sensitive pof1 mutants (pof1-6, pof1-12) show altered protein conformation at 36°C

  • Antibodies recognizing conformation-dependent epitopes may show different reactivity at various temperatures

  • Resolution: Test antibody performance at multiple temperatures

• Stress response conditions:

  • Cadmium exposure affects Zip1 levels and may alter Pof1 localization or modification

  • Cell cycle synchronization can affect F-box protein expression

  • Resolution: Control experimental timing carefully and include time-matched controls

• Fixation and extraction variables:

  • Crosslinking fixatives may mask epitopes in protein complexes

  • Detergent strength affects retention of membrane-associated pools

  • Resolution: Compare multiple fixation methods and extraction conditions

• Buffer system optimization:

  • pH sensitivity: Test pH range 6.8-8.0 to optimize epitope recognition

  • Salt concentration: Affects antibody-antigen interaction and non-specific binding

  • Resolution: Perform buffer optimization experiments for each application

Data-based example: When studying protein turnover, cycloheximide chase assays should be carefully controlled, as degradation kinetics can vary significantly between experimental conditions. In studies of Zip1, researchers quantified band intensity at regular intervals after cycloheximide addition, revealing differential stability of phosphorylated versus unphosphorylated forms .

How can high-throughput antibody development methods be applied to generate improved anti-SPBP16F5.05c antibodies?

Cutting-edge approaches for next-generation SPBP16F5.05c antibody development include:

• Single B cell screening technologies:

  • High-throughput scRNA-seq and VDJ sequencing of memory B cells can identify antigen-binding clonotypes

  • From 676 antigen-binding IgG1+ clonotypes, researchers have successfully identified antibodies with nanomolar affinity (KD ~10^-9 M)

  • This approach enables rapid screening of antibody candidates without traditional hybridoma generation

• Computational design and affinity maturation:

  • Structural antibody databases such as SAbDab provide templates for computational design

  • IsAb computational protocol integrates multiple Rosetta tools for antibody design

  • Machine learning and supercomputing approaches can predict beneficial mutations

• Collaborative approaches combining methods:

  • Integration of computational prediction with high-throughput experimental validation

  • Feedback loops incorporating experimental results into refined models

  • Cross-validation using multiple binding assays (ELISA, Biolayer Interferometry)

Research insight: Combining high-throughput antibody discovery with rigorous validation has yielded highly specific antibodies, as demonstrated in the development of Abs-9 against SpA5, which showed not only high affinity but also specificity confirmed by mass spectrometry .

What novel applications of anti-SPBP16F5.05c antibodies are emerging in research?

Innovative applications for SPBP16F5.05c antibodies in research include:

• Proteome-wide interaction mapping:

  • Antibodies can be used for immunoprecipitation followed by mass spectrometry

  • This approach has been successful in identifying novel substrates of F-box proteins

  • Application to SPBP16F5.05c could reveal previously unknown interaction partners

• Developmental timing studies:

  • Antibodies can track F-box protein expression during developmental transitions

  • Applications in studying the switch between proliferation and quiescence

  • Potential insights into stress response regulation during development

• Integration with single-cell technologies:

  • Combining antibody-based detection with single-cell RNA sequencing

  • Detecting protein levels alongside transcriptomic changes

  • Applications in heterogeneous cell populations

• Structural studies of protein complexes:

  • Antibodies can stabilize protein complexes for cryo-EM analysis

  • Fragment antibodies can facilitate crystallization of challenging proteins

  • Potential to resolve the structure of the complete SCFPof1 complex

Research potential: Studies of the SCFPof1 complex have already revealed its role in regulating the transcription factor Zip1 in response to cadmium . Expanding this approach could uncover additional substrates and regulatory mechanisms in different environmental conditions.

How might understanding SPBP16F5.05c function inform therapeutic antibody development strategies?

While SPBP16F5.05c is a yeast protein, insights from studying it can inform therapeutic antibody development in several ways:

• F-box protein targeting strategies:

  • Human F-box proteins play roles in cancer and other diseases

  • Structural studies of SPBP16F5.05c/Pof1 can inform epitope selection for human F-box proteins

  • Lessons from substrate recognition mechanisms can guide therapeutic development

• Methodology transfer:

  • Techniques optimized for SPBP16F5.05c antibodies can be applied to human targets

  • Validation strategies developed for research antibodies can improve therapeutic antibody quality

  • Cross-reactivity assessment methods are applicable to therapeutic development

• Fundamental biological insights:

  • Understanding ubiquitin-dependent proteolysis mechanisms is relevant across species

  • Cell cycle regulation principles discovered in yeast inform human disease mechanisms

  • Stress response pathways often have conserved elements