ppk18 Antibody

What is ppk18 and why is it studied in research?

ppk18 is a serine/threonine protein kinase found in Schizosaccharomyces pombe (fission yeast). It has been identified as a predicted serine/threonine protein kinase encoded by gene ID 3361535 . Fission yeast serves as an important model organism in molecular and cell biology research, with ppk18 being one of its approximately 4,970 protein-coding genes . Researchers study ppk18 to understand cellular signaling pathways and regulatory mechanisms in eukaryotic cells.

S. pombe has become a powerful model organism because its cells maintain their shape by growing exclusively through cell tips and divide by medial fission to produce two daughter cells of equal size, making it valuable for cell cycle research . Additionally, approximately 70% of S. pombe proteins have human orthologs, with over 1,500 associated with human disease, making research on proteins like ppk18 potentially relevant to human biology .

What are the common experimental applications for ppk18 antibody?

Based on available product information, ppk18 antibodies are primarily used in the following experimental applications:

• Western Blotting (WB): For detection and quantification of ppk18 protein in cell lysates

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative measurement of ppk18 in solution

Researchers typically use these applications to:

• Verify gene knockdown or overexpression

• Study protein expression under different conditions

• Investigate protein-protein interactions

• Analyze post-translational modifications

When designing experiments with ppk18 antibody, researchers should consider the appropriate sample preparation methods for S. pombe, which may differ from those used for mammalian cells.

What are the key specifications of commercially available ppk18 antibodies?

Based on available data, typical specifications for ppk18 antibodies include:

When selecting a ppk18 antibody, researchers should consider these specifications in relation to their specific experimental needs, particularly regarding the detection method, sensitivity requirements, and compatibility with other reagents in the experimental system.

How should ppk18 antibody be validated for specificity in S. pombe research?

Validating antibody specificity is crucial for reliable research outcomes. For ppk18 antibody, recommended validation approaches include:

• Western blot analysis using positive and negative controls:

  • Positive control: Wild-type S. pombe expressing ppk18

  • Negative control: ppk18 knockout/deletion strain

  • Expected result: A single band at the predicted molecular weight (~98 kDa) in positive control and absence in negative control

• Peptide competition assay:

  • Pre-incubate the antibody with excess immunizing peptide

  • Run parallel Western blots with treated and untreated antibody

  • Expected result: Signal reduction or elimination in the peptide-blocked sample

• Immunoprecipitation followed by mass spectrometry:

  • Use ppk18 antibody to pull down the protein from cell lysates

  • Analyze by mass spectrometry to confirm identity

  • Expected result: Identification of ppk18 peptides in the immunoprecipitated sample

• Correlation with RNA expression data:

  • Compare protein levels detected by the antibody with mRNA expression data

  • Expected result: Correlation between protein and mRNA levels across different conditions

These validation approaches help ensure that experimental findings genuinely reflect ppk18 biology rather than non-specific interactions.

How can researchers optimize protein extraction from S. pombe for ppk18 antibody applications?

Extracting proteins from S. pombe for antibody-based detection requires optimization due to the rigid cell wall of fission yeast. Based on established protocols in yeast research:

• Cell wall disruption methods comparison:

  • Glass bead homogenization: Effective but can generate heat

  • Enzymatic digestion with zymolyase: Gentler but may affect some proteins

  • Cryogenic grinding: Preserves most post-translational modifications but more laborious

• Recommended buffer composition:

  • Base buffer: 50 mM Tris-HCl pH 7.5, 150 mM NaCl

  • Protease inhibitors: Complete protease inhibitor cocktail

  • Phosphatase inhibitors (if studying phosphorylation): 5 mM NaF, 1 mM Na₃VO₄

  • Reducing agent: 1 mM DTT

  • Detergent: 0.5% NP-40 or 1% Triton X-100

• Extraction efficiency considerations:

  • Cell density: Mid-log phase cultures (OD₆₀₀ = 0.5-0.8) typically yield optimal results

  • Temperature: Keep samples cold throughout extraction to prevent protein degradation

  • Duration: Minimize processing time to preserve protein integrity

Research has shown that different extraction methods may yield varying results in terms of protein recovery, especially for kinases like ppk18 that may be present at relatively low abundance or have specific subcellular localizations .

What considerations are important when studying ppk18 phosphorylation states?

As ppk18 is a serine/threonine kinase, researchers often need to investigate its phosphorylation status:

• Phosphorylation-specific detection strategies:

  • Phos-tag SDS-PAGE: Can separate phosphorylated from non-phosphorylated forms

  • Phospho-specific antibodies: May be needed to detect specific phosphorylation sites

  • Lambda phosphatase treatment: Control samples to confirm phosphorylation specificity

• Sample preparation considerations:

  • Rapid sample processing: Minimize time between cell harvesting and protein denaturation

  • Phosphatase inhibitors: Include multiple types (e.g., NaF, Na₃VO₄, β-glycerophosphate)

  • Denaturation conditions: Boiling in SDS buffer immediately after cell lysis

• Controls for phosphorylation studies:

  • Untreated vs. phosphatase-treated samples

  • Cells under conditions known to alter kinase activity (e.g., nutrient starvation, cell cycle synchronization)

The MASTL/PP2A cell cycle kinase-phosphatase module study provides a relevant example of how phosphorylation states of kinases can be carefully analyzed in research settings .

How should researchers design experiments to study ppk18 interactions with other proteins?

To study protein-protein interactions involving ppk18, consider these methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Use ppk18 antibody to pull down protein complexes

  • Analyze associated proteins by Western blot or mass spectrometry

  • Controls: IgG control, lysate from ppk18-deleted strain

• Proximity-based labeling:

  • Express ppk18 fused to BioID or APEX2

  • Identify proteins in proximity through biotinylation and streptavidin pulldown

  • Advantage: Can detect transient or weak interactions

• Yeast two-hybrid screening:

  • Use ppk18 as bait to screen for interacting proteins

  • Verification with reciprocal tests and in vivo methods

  • Consideration: May identify indirect interactions

• Fluorescence microscopy approaches:

  • FRET (Förster Resonance Energy Transfer) for direct interactions

  • Co-localization studies with GFP-tagged ppk18 and potential interactors

  • Note that in 2006, subcellular localization of almost all S. pombe proteins was published using GFP tagging

When interpreting interaction data, researchers should consider the dynamic nature of kinase interactions, which may be influenced by cell cycle stage, stress conditions, or post-translational modifications.

What approaches can be used to study the functional consequences of ppk18 activity?

To investigate ppk18 kinase function in cellular processes:

• Genetic manipulation strategies:

  • Gene deletion/knockout: Study loss-of-function phenotypes

  • Point mutations: Create kinase-dead variants (e.g., mutations in catalytic domain)

  • Conditional expression: Use repressible/inducible promoters for temporal control

• Kinase activity assays:

  • In vitro kinase assays with recombinant protein

  • Phosphorylation site mapping using mass spectrometry

  • Phospho-specific antibodies to track substrate phosphorylation

• Phenotypic analysis methods:

  • Cell cycle progression analysis

  • Cellular stress response assessment

  • Morphological analysis

  • Growth rate determination under various conditions

• Comparative approaches:

  • Cross-species complementation experiments

  • Homology modeling based on related kinases

  • Evolutionary analysis across fungi and other eukaryotes

Recent studies on MASTL/Greatwall kinase (a related kinase in other systems) have shown how kinases can regulate critical cellular processes through phosphorylation-dependent signaling networks , providing methodological inspiration for ppk18 research.

What are common issues with ppk18 antibody detection and how can they be resolved?

Researchers may encounter several technical challenges when working with ppk18 antibody:

• Weak or no signal in Western blot:

  • Increase protein loading (up to 50-100 μg total protein)

  • Optimize antibody concentration (try 1:500 to 1:2000 dilutions)

  • Extend primary antibody incubation (overnight at 4°C)

  • Use enhanced chemiluminescence (ECL) substrate with longer exposure times

  • Consider more sensitive detection methods (e.g., SuperSignal West Femto)

• Multiple bands or high background:

  • Increase blocking time or concentration (5% BSA or milk)

  • Add 0.1-0.5% Tween-20 in wash buffer

  • Pre-absorb antibody with cell lysate from ppk18-deletion strain

  • Use freshly prepared samples to minimize degradation

  • Consider using more stringent washing conditions

• Inconsistent results between experiments:

  • Standardize protein extraction method

  • Use internal loading controls consistently

  • Maintain consistent cell growth conditions

  • Aliquot antibody to avoid freeze-thaw cycles

  • Document lot-to-lot variations in antibody performance

• Cross-reactivity with related proteins:

  • Verify specificity using knockout controls

  • Use computational analysis to identify potential cross-reactive proteins

  • Consider epitope mapping to understand antibody binding sites

Based on experience with yeast proteins, using freshly prepared samples and including appropriate controls is particularly important for consistent results .

How should researchers interpret ppk18 phosphorylation data in the context of cell cycle regulation?

Interpreting phosphorylation data requires careful consideration of biological context:

• Cell cycle synchronization considerations:

  • Different synchronization methods may affect phosphorylation patterns

  • Time-course experiments should include multiple time points

  • Single-cell analysis may complement population-based approaches

• Integration with known regulatory networks:

  • Compare with data on related kinases (e.g., MASTL/Greatwall in other organisms)

  • Consider connections to PP2A phosphatase networks

  • Examine potential links to cell cycle checkpoints

• Phosphorylation site analysis:

  • Quantitative analysis of site occupancy

  • Evolutionary conservation of phosphorylation sites

  • Structural implications of phosphorylation

• Functional validation approaches:

  • Phosphomimetic and phospho-deficient mutations

  • Temporal correlation with cellular events

  • Chemical genetics approaches (e.g., analog-sensitive kinase variants)

The phosphoregulation of kinases like MASTL by mTORC1, as described in search result , provides a model for how phosphorylation data can be interpreted in the context of regulatory networks.

How does ppk18 in S. pombe compare to related kinases in other organisms?

Understanding evolutionary relationships can provide insight into ppk18 function:

• Orthology and homology analysis:

  • ppk18 belongs to the AGC family of serine/threonine kinases

  • Related kinases include MASTL/Greatwall in vertebrates

  • Rim15 in Saccharomyces cerevisiae shares functional similarities

• Functional conservation assessment:

  • Core catalytic domains show higher conservation

  • Regulatory regions often show greater divergence

  • Substrate specificity may vary across species

• Experimental approaches for comparative studies:

  • Cross-species complementation experiments

  • Heterologous expression of ppk18 in other systems

  • Comparative phosphoproteomics

• Evolutionary trajectory analysis:

  • Presence/absence across fungal lineages

  • Rate of sequence evolution compared to other kinases

  • Correlation with emergence of specific cellular processes

The genetic diversity of S. pombe strains is slightly less than that of budding yeast, which may affect the evolutionary context of proteins like ppk18 .

How can cross-species antibody reactivity be evaluated for ppk18 research?

When considering using antibodies across related species:

• Epitope sequence alignment analysis:

  • Compare immunogen sequence with potential cross-reactive species

  • Look for regions of high conservation

  • Predict potential cross-reactivity based on sequence identity

• Experimental validation approaches:

  • Western blot analysis with lysates from multiple species

  • Include positive and negative controls for each species

  • Titrate antibody concentration to optimize signal-to-noise ratio

• Considerations for S. pombe-specific research:

  • S. pombe diverged from S. cerevisiae approximately 1,100 million years ago

  • Despite sharing ~3,600 orthologous proteins, antibody cross-reactivity cannot be assumed

  • Species-specific post-translational modifications may affect epitope recognition

• Documentation requirements:

  • Clearly report validation steps for each species tested

  • Note any differences in optimal conditions between species

  • Specify the exact strain used for validation (e.g., strain 972 / ATCC 24843)

Understanding the biodiversity and evolutionary history of S. pombe strains, as described in search result , is important when evaluating cross-species reactivity.

How can proteomics approaches enhance ppk18 antibody-based research?

Integrating antibody-based detection with proteomics offers several advantages:

• Mass spectrometry-based identification strategies:

  • Immunoprecipitation coupled with LC-MS/MS

  • SILAC or TMT labeling for quantitative analysis

  • Phosphoproteomic analysis to identify substrates

• Proteome-wide interaction mapping:

  • Proximity-dependent biotinylation (BioID, APEX)

  • Affinity purification-mass spectrometry (AP-MS)

  • Cross-linking mass spectrometry (XL-MS) for structural insights

• Integration with S. pombe proteome resources:

  • Comparative proteomic and transcriptomic profiling data is available for S. pombe

  • Approximately 29.5% of the predicted S. pombe proteome has been identified through multidimensional biochemical prefractionation and LC ESI MS/MS

  • Analysis of protein abundance revealed that essential proteins are considerably more abundant (median ASC=12.6) than non-essential proteins (ASC=7.5)

• Data analysis considerations:

  • False discovery rate control in MS-based identification

  • Network analysis of interaction partners

  • Correlation with transcriptomic data for integrated analysis

The extensive proteomic analysis of S. pombe described in search result provides a valuable resource and methodological framework for integrating ppk18 research with wider proteome studies.

What considerations are important when designing CRISPR-based experiments to study ppk18 function?

CRISPR/Cas9 approaches for studying ppk18 in S. pombe require special considerations:

• Guide RNA design parameters:

  • S. pombe-optimized CRISPR systems may differ from those for mammalian cells

  • Target selection should consider S. pombe genome structure and PAM requirements

  • Off-target prediction using S. pombe genome databases

• Delivery and expression methods:

  • Plasmid-based expression vs. RNP delivery

  • Promoter selection for Cas9 and gRNA expression

  • Selection markers appropriate for S. pombe

• Repair template design considerations:

  • Homology-directed repair efficiency in S. pombe

  • Homology arm length optimization

  • Integration of epitope tags for antibody detection

• Validation approaches:

  • PCR-based genotyping strategies

  • Sequencing confirmation of edits

  • Antibody-based verification of protein modification/deletion

  • Phenotypic analysis to confirm functional effects

When interpreting CRISPR-edited cell phenotypes, researchers should consider potential adaptation or compensation mechanisms that may occur after ppk18 modification.

What emerging technologies might enhance ppk18 antibody applications in research?

Several emerging technologies hold promise for advancing ppk18 research:

• Single-cell proteomics approaches:

  • Mass cytometry (CyTOF) with metal-conjugated antibodies

  • Microfluidic-based single-cell Western blotting

  • Single-cell proteogenomic correlation

• Advanced imaging techniques:

  • Super-resolution microscopy for precise localization

  • Live-cell imaging with split-fluorescent protein complementation

  • Lattice light-sheet microscopy for dynamic studies

• Synthetic biology approaches:

  • Engineered allosteric switches for kinase activity control

  • Optogenetic regulation of kinase function

  • Biosensors for real-time monitoring of kinase activity

• Computational prediction and modeling:

  • Machine learning for interaction prediction

  • Molecular dynamics simulations of kinase regulation

  • Systems biology modeling of signaling networks

These technologies could provide unprecedented insight into ppk18 function, localization, and dynamics in live cells, potentially revealing novel aspects of its biology that are not accessible with current methods.

How might ppk18 research contribute to understanding conserved kinase regulation mechanisms?

Studies of ppk18 in S. pombe may broadly impact understanding of kinase regulation:

• Fundamental mechanisms of kinase regulation:

  • Activation loop phosphorylation dynamics

  • Allosteric regulation mechanisms

  • Protein-protein interaction networks in kinase control

• Cell cycle control mechanisms:

  • Checkpoint regulation by kinase-phosphatase modules

  • Timing mechanisms in cell cycle progression

  • Integration of nutrient sensing with cell cycle control

• Evolution of regulatory networks:

  • Conservation of kinase-substrate relationships

  • Divergence of regulatory mechanisms

  • Emergence of specialized functions in different lineages

• Translational relevance:

  • Insights into related human kinases

  • Potential therapeutic targets in diseases involving dysregulated kinase activity

  • Model for understanding kinase inhibitor mechanisms