PBL17 Antibody

What is PBL17 and why is it significant in plant research?

PBL17 (PBS1-Like kinase 17) is a receptor-like cytoplasmic kinase found in Arabidopsis thaliana that plays roles in plant immunity signaling networks. The protein is associated with Uniprot accession number Q8H1E3 and functions within phosphorylation cascades potentially related to pathogen recognition and response mechanisms. Understanding PBL17's function contributes to our broader knowledge of plant immune system signaling and may provide insights into engineering enhanced disease resistance in agricultural applications. The antibody against this protein enables researchers to detect, quantify, and localize PBL17 in experimental systems to elucidate its biological roles and regulation .

What are the validated applications for PBL17 antibody?

The PBL17 antibody has been validated for enzyme-linked immunosorbent assay (ELISA) and Western blotting (WB) applications. These techniques allow for both qualitative and quantitative assessment of PBL17 protein expression and modifications in experimental contexts. The antibody enables identification of the target antigen through these methods, facilitating research into protein expression patterns, post-translational modifications, and protein-protein interactions. For optimal experimental design, researchers should consider that this antibody was raised against recombinant Arabidopsis thaliana PBL17 protein and has confirmed reactivity with Arabidopsis thaliana samples .

What is the difference between using PBL17 antibody in ELISA versus Western blotting applications?

When using PBL17 antibody for ELISA, researchers can quantitatively measure the presence of PBL17 protein in solution, which is particularly useful for determining relative protein concentrations across different samples or treatment conditions. The antibody binds to the target protein immobilized on a plate surface, and detection systems reveal the relative abundance. In contrast, Western blotting with PBL17 antibody allows visualization of the protein after separation by molecular weight via gel electrophoresis, providing information about protein size, potential degradation products, post-translational modifications, and relative expression levels. Western blotting is preferable when confirming protein identity or investigating potential isoforms, while ELISA offers advantages in high-throughput quantitative analysis of native protein conformations .

What are the optimal storage and handling conditions for PBL17 antibody?

The PBL17 antibody requires careful storage and handling to maintain its immunoreactivity and specificity. Upon receipt, the antibody should be stored at either -20°C or -80°C, with the latter being preferred for long-term storage to minimize freeze-thaw cycles that can degrade antibody performance. For working solutions, the antibody is provided in a protective buffer containing 0.03% Proclin 300 (as a preservative), 50% glycerol, and 0.01M PBS at pH 7.4. This formulation enhances stability during freeze-thaw transitions. Researchers should aliquot the antibody into volumes appropriate for single experiments to avoid repeated freeze-thaw cycles, which significantly reduce antibody efficiency and specificity. When handling, maintain the antibody on ice during experimental procedures and avoid exposing it to direct light or extreme pH conditions .

How should researchers optimize Western blotting protocols for PBL17 detection?

For optimal Western blotting results with PBL17 antibody, researchers should consider several methodological adaptations. Begin with proper sample preparation by extracting proteins from Arabidopsis thaliana tissues using a buffer that preserves phosphorylation states if post-translational modifications are of interest. Load 20-50 μg of total protein per lane and separate using 10-12% SDS-PAGE gels. For transfer, PVDF membranes are recommended due to their higher protein binding capacity compared to nitrocellulose. Block membranes with 5% non-fat dry milk or bovine serum albumin in TBST (Tris-buffered saline with 0.1% Tween-20) for 1 hour at room temperature. Dilute the PBL17 antibody at 1:500 to 1:2000 in blocking buffer (specific optimization may be necessary) and incubate overnight at 4°C. After washing, use an appropriate anti-rabbit IgG secondary antibody conjugated to HRP. Because this antibody is polyclonal, validation with knockout/knockdown controls is strongly recommended to confirm specificity .

What are common causes of false positives or negatives when using PBL17 antibody, and how can researchers address them?

When working with PBL17 antibody, researchers may encounter false positives due to several factors: cross-reactivity with structurally similar plant kinases, particularly other PBS1-like family members; non-specific binding to highly abundant proteins; or secondary antibody binding to endogenous plant immunoglobulins. To mitigate these issues, researchers should: implement stringent blocking procedures using 5% BSA; include competitive blocking with the immunizing peptide if available; and validate results with genetic controls (PBL17 knockout/knockdown lines).

False negatives commonly result from: suboptimal protein extraction methods that fail to solubilize membrane-associated kinases; protein degradation during sample preparation; or epitope masking due to protein-protein interactions or post-translational modifications. To address these challenges: incorporate protease and phosphatase inhibitors in extraction buffers; optimize extraction conditions with different detergents (0.1-1% NP-40, Triton X-100, or CHAPS); and consider native versus denaturing conditions depending on the application. Additionally, researchers should titrate antibody concentrations and adjust incubation times to optimize signal-to-noise ratios for their specific experimental conditions .

How should researchers interpret differences in PBL17 detection patterns between various plant tissues and experimental conditions?

Interpreting variations in PBL17 detection across different plant tissues and experimental conditions requires systematic analysis of multiple factors. Different tissues may display varying PBL17 expression levels due to tissue-specific promoter activity, protein stability differences, or tissue-specific post-translational modifications that affect antibody recognition. When comparing patterns between conditions (e.g., pathogen-challenged versus control plants), researchers should normalize PBL17 signals to appropriate loading controls—household proteins with similar molecular weights but different biological functions than PBL17 are ideal to avoid overlapping regulatory mechanisms.

For methodologically sound interpretation, researchers should consider the following approach: (1) establish baseline expression patterns across tissues under standard conditions; (2) compare relative expression changes rather than absolute signals across different tissues; (3) verify unusual patterns with alternative detection methods such as RT-PCR for transcript levels; and (4) consider that absence of signal may represent expression below detection threshold rather than complete absence of the protein. Finally, remember that epitope accessibility may vary between experimental conditions, particularly if treatments induce changes in protein complex formation or subcellular localization, potentially affecting antibody binding efficacy independently of actual protein abundance .

How can researchers validate specificity when unexpected molecular weight bands appear in Western blots using PBL17 antibody?

When unexpected bands appear in Western blots with PBL17 antibody, researchers must systematically validate antibody specificity to distinguish between true PBL17-related signals and artifacts. The expected molecular weight of PBL17 should be compared with observed bands, considering that post-translational modifications, particularly phosphorylation events common in kinases, can cause mobility shifts. For comprehensive validation, researchers should implement multiple strategies: (1) perform peptide competition assays where pre-incubation of the antibody with excess immunizing peptide should eliminate specific bands; (2) analyze samples from PBL17 knockout/knockdown plants, which should show reduced or absent signal for specific bands; (3) employ recombinant PBL17 protein as a positive control with defined molecular weight; and (4) consider whether unexpected bands might represent splice variants, degradation products, or post-translationally modified forms of PBL17.

For bands suspected to be modified forms of PBL17, researchers can treat samples with phosphatases, glycosidases, or other modification-removing enzymes to observe whether band patterns simplify to the expected molecular weight. Additionally, mass spectrometry analysis of excised gel bands can provide definitive identification. These comprehensive validation approaches ensure reliable interpretation of Western blot results and prevent misattribution of non-specific signals to PBL17-related biology .

How can PBL17 antibody be utilized in investigating plant immune response pathways?

The PBL17 antibody represents a valuable tool for dissecting plant immune signaling networks, particularly in examining how receptor-like cytoplasmic kinases integrate and transmit signals from pattern recognition receptors. Researchers can implement several sophisticated approaches using this antibody: (1) temporal profiling of PBL17 expression and phosphorylation status during pathogen challenge using time-course immunoblotting; (2) spatial analysis of PBL17 redistribution during immune responses through subcellular fractionation followed by immunoblotting or immunofluorescence microscopy; and (3) identification of PBL17-associated protein complexes before and after immune elicitation using antibody-based pull-downs coupled with mass spectrometry.

Additionally, the antibody can facilitate investigation of post-translational modification dynamics, particularly phosphorylation events that likely regulate PBL17 activity. Researchers might employ phospho-specific antibody screening after PBL17 immunoprecipitation or develop phospho-specific antibodies for key regulatory sites identified through phosphoproteomic analysis. These approaches could reveal how PBL17 contributes to signal transduction cascades during plant-pathogen interactions, potentially identifying novel intervention points for enhancing crop disease resistance .

What methodological adaptations are needed to apply PBL17 antibody in super-resolution microscopy techniques?

Adapting PBL17 antibody for super-resolution microscopy techniques requires specific methodological modifications to achieve optimal spatial resolution while maintaining signal specificity. For techniques like Structured Illumination Microscopy (SIM), Stimulated Emission Depletion (STED), or Single-Molecule Localization Microscopy (SMLM), researchers should consider: (1) using directly labeled primary antibodies to minimize the distance between fluorophore and target, reducing the "linkage error" that limits resolution; (2) optimizing fixation protocols to preserve cellular ultrastructure while maintaining epitope accessibility—paraformaldehyde fixation (3-4%) followed by minimal permeabilization is often suitable for maintaining membrane-associated structures where signaling kinases might localize; and (3) implementing specific blocking strategies to minimize non-specific binding, which becomes more problematic as resolution increases.

For multi-color super-resolution imaging to co-localize PBL17 with interaction partners or cellular structures, researchers should carefully select secondary antibody combinations to minimize spectral overlap and employ appropriate controls to distinguish between specific signal and background autofluorescence, which is particularly challenging in plant tissues. Additionally, sample preparation should include steps to reduce plant tissue autofluorescence, such as brief treatment with sodium borohydride or extended incubation in Sudan Black B. These technical adaptations will enable visualization of PBL17's subcellular distribution at nanoscale resolution, potentially revealing previously undetectable spatial organization relevant to its signaling functions .