PBL23 Antibody

What is PBL23 Antibody and what organism does it target?

PBL23 Antibody (CSB-PA599770XA01DOA) is a polyclonal antibody that specifically targets the PBL23 protein in Arabidopsis thaliana (Mouse-ear cress), a model plant organism widely used in molecular biology research. It is raised in rabbit using recombinant Arabidopsis thaliana PBL23 protein as the immunogen and is purified using antigen affinity chromatography .

The antibody has the following specifications:

What are the validated applications for PBL23 Antibody?

PBL23 Antibody has been validated for use in ELISA and Western Blot applications, with Western Blot being particularly useful for ensuring the identification of the antigen . Based on general antibody application principles, researchers should consider the following methodological guidelines:

For Western Blot applications:

• Sample preparation: Typical protein extraction from Arabidopsis tissues should be performed using appropriate lysis buffers (similar to those containing DTT, PMSF, lysozyme, and Triton X-100 used for other plant proteins) .

• Sample loading: Use appropriate protein concentration (typically 20-50 μg of total protein).

• Detection: A secondary antibody conjugated to HRP or other detection systems is required.

For ELISA applications:

• Coating: Use purified recombinant PBL23 protein for standard curve generation.

• Blocking: BSA or casein-based blocking solutions (typically 1-5%) are recommended.

• Dilution ranges: Titration is recommended for optimal results.

How should I optimize PBL23 Antibody concentration for Western Blotting?

Optimizing antibody concentration is critical for balancing specific signal detection with minimizing background. For PBL23 Antibody, follow this methodological approach:

• Initial titration experiment:

  • Prepare identical membrane strips containing your target protein

  • Test a dilution series (e.g., 1:500, 1:1000, 1:2000, 1:5000, 1:10000)

  • Select the dilution that provides the best signal-to-noise ratio

• Validation controls:

  • Positive control: Recombinant PBL23 protein

  • Negative control: Lysate from organisms not expressing PBL23

  • Technical control: Omission of primary antibody

• Optimization variables:

  • Incubation time (1-16 hours)

  • Incubation temperature (4°C, room temperature)

  • Blocking reagent (5% milk, 3-5% BSA)

  • Washing stringency (TBST concentration, wash duration)

This approach aligns with standard practices for antibody optimization that yield reliable data for publication-quality research, similar to methods used for other antibodies such as PRL-3 .

How do I validate the specificity of PBL23 Antibody for my experiments?

Validation of antibody specificity is critical for ensuring experimental rigor. For PBL23 Antibody, implement the following validation strategy:

• Genetic validation:

  • Test the antibody in PBL23 knockout or knockdown plant lines

  • Expected result: Significant reduction or absence of signal in knockout/knockdown lines

• Recombinant protein validation:

  • Express and purify recombinant PBL23 protein

  • Run Western blot with increasing amounts (e.g., 0.1-1.0 μg)

  • Expected result: Linear increase in signal intensity

• Competitive binding assay:

  • Pre-incubate antibody with excess purified antigen before application

  • Expected result: Significant reduction in signal

• Cross-reactivity assessment:

  • Test antibody against related proteins (e.g., other PBL family members)

  • Expected result: No cross-reactivity with homologous proteins

These validation methods are based on established protocols for antibody validation used in recent studies with other antibodies, which demonstrated that thorough validation is essential for reliable research outcomes .

How can I use PBL23 Antibody for studying plant immune responses?

PBL23 belongs to a family of proteins involved in plant immunity signaling. To investigate its role in immune responses:

• Immunoprecipitation coupled with mass spectrometry:

  • Use PBL23 Antibody to pull down PBL23 and associated proteins

  • Identify interaction partners through LC-MS/MS

  • Map the interactome changes during immune challenge

• Co-immunoprecipitation for specific interactions:

  • Investigate interactions with known immunity proteins

  • Compare interactions in healthy vs. pathogen-challenged plants

  • Validate with reciprocal co-IP experiments

• Phosphorylation state analysis:

  • Use the antibody to immunoprecipitate PBL23 before and after pathogen treatment

  • Analyze phosphorylation status by phospho-specific Western blotting or mass spectrometry

  • Correlate phosphorylation changes with immune signaling events

• Immunohistochemistry for localization studies:

  • Optimize fixation for plant tissues (typically 4% paraformaldehyde)

  • Use antigen retrieval methods if necessary

  • Employ fluorescent secondary antibodies for co-localization studies

This approach draws on established methodologies used for studying other plant immunity proteins and can be adapted for PBL23 research .

What methodologies can be used to study PBL23 protein-protein interactions in planta?

Investigating protein-protein interactions in the native plant context requires specialized approaches:

• Bimolecular Fluorescence Complementation (BiFC):

  • Clone PBL23 into BiFC vectors

  • Use PBL23 Antibody to validate expression levels in parallel experiments

  • Analyze interaction-dependent fluorescence reconstitution

• Förster Resonance Energy Transfer (FRET):

  • Tag PBL23 and potential interactors with appropriate fluorophores

  • Use PBL23 Antibody to confirm expression levels

  • Measure energy transfer as indication of protein proximity

• Proximity Ligation Assay (PLA):

  • Utilize PBL23 Antibody with antibodies against candidate interactors

  • Secondary antibodies with oligonucleotide probes enable amplification of signal when proteins are in close proximity

  • Visualize interactions in situ with cellular resolution

• Co-immunoprecipitation from plant tissues:

  • Extract proteins under native conditions

  • Immunoprecipitate with PBL23 Antibody

  • Identify interacting partners by Western blot or mass spectrometry

These methods provide complementary approaches to validate interactions, similar to strategies used in studying complex signaling networks in other research contexts .

Why might I observe multiple bands when using PBL23 Antibody in Western blot?

Multiple bands in Western blots using PBL23 Antibody could have several methodological explanations:

• Post-translational modifications:

  • Phosphorylation, glycosylation, or ubiquitination can alter protein mobility

  • Verification approach: Treat samples with appropriate enzymes (phosphatases, glycosidases) before Western blot

• Protein degradation:

  • Improper sample handling leading to proteolytic cleavage

  • Solution: Add protease inhibitor cocktail to extraction buffer and maintain samples at 4°C

• Splice variants:

  • Alternative splicing producing different isoforms

  • Verification: RT-PCR analysis of transcript variants

• Cross-reactivity:

  • Antibody recognizing homologous proteins

  • Validation: Test specificity using recombinant proteins of related family members

• Non-specific binding:

  • Inadequate blocking or washing

  • Optimization: Increase blocking agent concentration (3-5% BSA) and washing stringency

The resolution approach should be methodical, addressing each potential cause systematically while maintaining appropriate controls .

How can I overcome weak or absent signal when using PBL23 Antibody?

When facing weak or absent signals with PBL23 Antibody, implement this systematic troubleshooting approach:

• Sample preparation optimization:

  • Enrich target protein by subcellular fractionation

  • Use gentler extraction methods to preserve epitopes

  • Ensure adequate protein concentration (verify by Ponceau S staining)

• Antibody incubation conditions:

  • Increase antibody concentration incrementally

  • Extend incubation time (overnight at 4°C)

  • Add 0.1% Tween-20 to reduce non-specific binding

• Detection system enhancement:

  • Switch to more sensitive detection systems (e.g., enhanced chemiluminescence)

  • Use signal amplification methods (e.g., tyramide signal amplification)

  • Try biotin-streptavidin systems for increased sensitivity

• Epitope accessibility improvement:

  • For denatured epitopes: Use different reducing agents or denaturing conditions

  • For conformational epitopes: Try native gel electrophoresis

• Storage and handling assessment:

  • Check antibody storage conditions (avoid repeated freeze-thaw cycles)

  • Verify buffer compatibility with application

  • Consider antibody degradation (age, improper storage)

This methodical approach has proven effective for troubleshooting similar antibodies in research contexts .

How does PBL23 Antibody compare with other plant immunity-related antibodies for research applications?

When selecting antibodies for plant immunity research, consider these comparative factors:

For comprehensive immunity studies, researchers often employ multiple antibodies to track signaling cascades. When using PBL23 Antibody, complement with other immunity markers to establish pathway connections and functional relationships .

What are the cutting-edge applications of antibodies like PBL23 in plant immunity research?

Recent advances in antibody applications for plant immunity research include:

• Single-cell protein analysis:

  • Using antibodies with microfluidic systems for cell-specific protein quantification

  • Application: Tracking cell-type specific PBL23 expression during infection

• ChIP-seq for transcription factor studies:

  • If PBL23 functions in transcriptional regulation, ChIP-grade antibodies enable genome-wide binding site identification

  • Methodology: Optimize crosslinking conditions specifically for plant tissues (1-3% formaldehyde)

• Antibody-based biosensors:

  • Immobilizing PBL23 Antibody on nanomaterials for real-time detection

  • Application: Monitoring immune responses in living plant tissues

• CRISPR-directed antibody recruitment:

  • Using dCas9-antibody fusions to recruit proteins to specific genomic loci

  • Potential: Studying the effect of PBL23 localization on chromatin structure

• Multiplexed antibody imaging:

  • Combining PBL23 Antibody with antibodies against other immunity proteins

  • Technology: Mass cytometry or multiplexed immunofluorescence for comprehensive pathway analysis

These approaches represent the cutting edge of plant immunity research and demonstrate how antibodies continue to enable new experimental paradigms .

How should I prepare plant samples for optimal PBL23 Antibody detection?

Sample preparation is critical for successful antibody detection in plant tissues:

• Protein extraction protocols:

  • For total protein: Grind tissue in liquid nitrogen and extract with buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM DTT, and protease inhibitor cocktail

  • For membrane-associated proteins: Use buffer with 1% digitonin or 0.5% NP-40

  • For nuclear proteins: Include nuclear isolation step before protein extraction

• Tissue-specific considerations:

  • Young tissues: Generally higher protein yield, less interfering compounds

  • Leaves: Remove waxy cuticle with brief detergent treatment

  • Roots: Wash thoroughly to remove soil contaminants

  • Seeds: Use stronger extraction buffer with higher detergent concentration

• Protein quantification:

  • Use Bradford or BCA assay compatible with extraction buffer

  • Normalize loading precisely (20-50 μg total protein per lane)

• Sample storage:

  • Add glycerol to 10% final concentration before freezing

  • Store extracted proteins at -80°C in single-use aliquots

  • Avoid repeated freeze-thaw cycles

These methodologies are based on established protocols for plant protein extraction and have been validated for detection of low-abundance signaling proteins .

What controls should be included when using PBL23 Antibody for developmental studies?

Robust experimental design requires appropriate controls for developmental studies:

• Essential negative controls:

  • Genetic: pbl23 knockout/knockdown lines

  • Antibody: Primary antibody omission

  • Developmental: Tissues known not to express PBL23

  • Technical: Pre-immune serum at equivalent concentration

• Positive controls:

  • Recombinant PBL23 protein (spiked into negative samples)

  • Tissues with confirmed high PBL23 expression

  • Transgenic lines overexpressing PBL23

• Expression normalization controls:

  • Housekeeping proteins (e.g., actin, tubulin, GAPDH)

  • Total protein measurement (Ponceau S, Coomassie)

  • Tissue-specific markers for mixed tissue samples

• Developmental stage verification:

  • Morphological markers

  • Stage-specific marker proteins

  • Documentation of growth conditions and developmental timing

• Experimental design considerations:

  • Biological replicates (minimum n=3)

  • Technical replicates for each biological sample

  • Randomization of sample processing order

These controls ensure that observed changes in PBL23 levels are genuinely related to developmental transitions rather than technical artifacts or natural variation .

How should I quantify and analyze Western blot data using PBL23 Antibody?

Proper quantification of Western blot data requires rigorous methodology:

• Image acquisition optimization:

  • Capture images within the linear dynamic range of your detection system

  • Avoid pixel saturation (verify with exposure series)

  • Use consistent acquisition parameters between samples

• Quantification approach:

  • Use image analysis software (ImageJ, Image Lab, etc.)

  • Define measurement area consistently for all samples

  • Subtract local background for each lane

• Normalization strategies:

  • Normalize to loading control (housekeeping protein)

  • Alternative: Total protein normalization using Ponceau S or Coomassie

  • For phosphorylation studies: Normalize phospho-signal to total protein signal

• Statistical analysis:

  • For multiple comparisons: ANOVA followed by appropriate post-hoc test

  • For comparing two conditions: t-test or non-parametric equivalent

  • Report exact p-values and effect sizes, not just significance

• Data presentation:

  • Include representative blot images showing all experimental conditions

  • Present quantified data as mean ± standard deviation or SEM

  • Use appropriate scale bars that don't exaggerate differences

This methodological approach ensures robust, reproducible quantification of Western blot data and has been validated in numerous plant immunity studies .

How can I reconcile contradictory results when using PBL23 Antibody across different experimental platforms?

When facing contradictory results between experimental platforms, implement this systematic reconciliation approach:

• Technical validation:

  • Confirm antibody lot consistency

  • Verify protein extraction efficiency across platforms

  • Assess platform-specific technical limitations

• Biological context assessment:

  • Evaluate differences in growth conditions

  • Consider developmental stage variations

  • Examine genetic background differences

• Epitope accessibility analysis:

  • Different techniques may expose/mask epitopes differently

  • Test native vs. denatured conditions

  • Consider post-translational modifications affecting epitope recognition

• Orthogonal validation:

  • Complement antibody-based detection with transcript analysis

  • Use genetic approaches (mutation, overexpression)

  • Apply fluorescent protein tagging for in vivo validation

• Resolution strategies:

  • Implement multiple antibodies targeting different epitopes

  • Develop standardized protocols across platforms

  • Consider platform-specific calibration with recombinant standards

This methodological framework provides a path to resolve contradictions and establish confidence in experimental observations, similar to approaches used when reconciling contradictory findings in other antibody-based research .

How might computational antibody design improve future iterations of PBL23 Antibody?

Computational approaches are revolutionizing antibody development:

• Epitope optimization:

  • In silico prediction of immunogenic epitopes specific to PBL23

  • Structure-based epitope design to target functionally relevant domains

  • Algorithms to minimize cross-reactivity with related proteins

• Affinity maturation simulation:

  • Computational modeling of antibody-antigen interactions

  • Virtual screening of affinity-enhancing mutations

  • Molecular dynamics simulations to predict binding kinetics

• Developability profiling:

  • Prediction of physicochemical properties affecting stability

  • Identification of potential post-translational modification sites

  • Assessment of aggregation propensity

• Machine learning applications:

  • Training models on existing antibody datasets

  • Predicting optimal framework regions for plant protein recognition

  • Designing multi-specific antibodies for complex signaling studies

These computational approaches could lead to next-generation PBL23 antibodies with enhanced specificity, sensitivity, and application versatility, similar to advances being made in therapeutic antibody development .

What emerging technologies might enhance the utility of PBL23 Antibody for plant immunity research?

Emerging technologies poised to transform antibody applications in plant research include:

• Nanobody development:

  • Single-domain antibodies derived from camelid antibodies

  • Advantages: Smaller size, increased tissue penetration, stability

  • Application: Intracellular tracking of PBL23 in living plant cells

• Antibody-enzyme fusion proteins:

  • Direct coupling of enzymatic reporters to PBL23 antibodies

  • Benefits: Signal amplification, reduced protocol complexity

  • Example: Peroxidase-antibody fusions for one-step detection

• Proximity-dependent labeling:

  • Antibody-APEX/BioID fusions for proximity proteomics

  • Application: Mapping the PBL23 protein interaction network in situ

  • Advantage: Captures transient interactions in native cellular environment

• Microfluidic antibody arrays:

  • High-throughput analysis of multiple immunity components

  • Application: Simultaneous profiling of PBL23 with other immunity proteins

  • Benefit: Temporal resolution of signaling cascades

• CRISPR-based antibody alternatives:

  • Programmable binding proteins as alternatives to traditional antibodies

  • Application: Visualizing endogenous PBL23 through CRISPR-display technologies

  • Advantage: Genetic encoding for in vivo experiments

These technologies represent the frontier of plant immunity research tools and may overcome current limitations in studying dynamic immune signaling processes .