PBL21 Antibody
Description
Biological Role of the Target Protein
The Q9LDZ5 protein belongs to the proteome of Arabidopsis thaliana, a species used extensively to study plant development, stress responses, and cellular signaling. While specific functional data for Q9LDZ5 is limited, polyclonal antibodies like PBL21 are designed to recognize epitopes on this protein, enabling researchers to study its localization, expression patterns, and interactions .
Applications in Research
Polyclonal antibodies such as PBL21 are valued for their broad epitope recognition, making them versatile tools in:
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Western blotting (WB): Detecting protein expression levels .
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Immunofluorescence (IF): Localizing proteins within tissues .
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Immunoprecipitation (IP): Isolating protein complexes for downstream analysis .
Their heterogeneity allows them to bind multiple epitopes of the same antigen, increasing assay sensitivity compared to monoclonal antibodies (mAbs) .
Key Validation Methods
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Western Blot (WB): Assesses antibody specificity by detecting target protein bands in lysates .
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Immunofluorescence (IF): Evaluates cellular localization using fluorescence microscopy .
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Knockout (KO) Cell Lines: Gold-standard controls to confirm antibody specificity .
Challenges in Polyclonal Antibodies
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Batch-to-Batch Variability: Sourced from pooled B-cell lineages, pAbs can exhibit inconsistent performance across lots .
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Cross-Reactivity: Potential binding to non-target proteins .
Industry Trends
Recent studies (e.g., Ayoubi et al., 2023) highlight the importance of validating antibodies using KO lines, with recombinant antibodies outperforming polyclonal and monoclonal variants in assays . While PBL21’s validation data is not explicitly published, such methodologies are critical for ensuring its reliability .
Comparative Analysis with Other Antibodies
| Feature | PBL21 Antibody | Recombinant Antibodies | Monoclonal Antibodies |
|---|---|---|---|
| Epitope Diversity | Broad (multiple epitopes) | Broad (engineered mix) | Single epitope |
| Production | Animal-derived | Recombinant | Hybridoma-derived |
| Reproducibility | Variable (batch-dependent) | Consistent | Consistent |
| Cost | Moderate | High | Moderate–High |
Polyclonal antibodies like PBL21 remain cost-effective for exploratory research, but recombinant alternatives offer superior reproducibility .
Future Directions
Emerging technologies, such as antibody sequencing and recombinant production, could mitigate variability in polyclonal antibodies. For instance, sequencing PBL21’s epitope-binding regions could enable its recombinant synthesis, ensuring long-term consistency .
Product Specs
Constituents: 50% Glycerol, 0.01M Phosphate Buffered Saline (PBS), pH 7.4
Target Background
Q&A
What is PBLD antibody and what epitopes does it target?
PBLD antibody targets Phenazine Biosynthesis-Like Protein Domain Containing 1 (PBLD1), a human protein with a molecular weight of approximately 31.6 kDa . These antibodies are available in various formats, with the most common being monoclonal antibodies raised in mouse using full-length recombinant human PBLD protein (NP_071412) produced in HEK293T cells . The clone 4F6 is a frequently referenced monoclonal variant that targets specific epitopes of the PBLD protein .
| Characteristics | Details |
|---|---|
| Full Name | Phenazine Biosynthesis-Like Protein Domain Containing 1 |
| Molecular Weight | 31.6 kDa |
| UniProt ID | P30039 |
| NCBI Reference | NM_022129 |
| Common Epitope Regions | Full-length protein, N-terminal, C-terminal |
The PBLD protein contains several distinct domains that can serve as epitopes for antibody binding. Researchers should select antibodies targeting epitopes relevant to their experimental design and research questions.
What are the validated applications for PBLD antibodies in research?
PBLD antibodies have been validated for multiple research applications, with varying degrees of optimization depending on the specific antibody clone and format:
When selecting a PBLD antibody for your research, consider the specific application requirements and choose an antibody that has been validated for your intended experimental design. Western blotting appears to be the most widely validated application across different PBLD antibody products .
What species reactivity is observed with PBLD antibodies?
The reactivity profile of PBLD antibodies varies between products:
| Antibody Type | Species Reactivity | Reference |
|---|---|---|
| Mouse monoclonal (OTI4F6) | Human | |
| Mouse monoclonal (4F6) | Human | |
| Rabbit polyclonal variants | Human, Mouse, Rat | |
| Extended reactivity variants | Human, Cow, Horse |
Species cross-reactivity should be experimentally validated for your specific samples, as sequence homology does not always predict antibody binding. For comparative studies across species, select an antibody with demonstrated multi-species reactivity or validate the cross-reactivity experimentally.
How can I validate the specificity of PBLD antibodies for my experimental design?
Antibody validation is critical for ensuring reliable research results. Recent studies indicate that approximately 50% of commercial antibodies fail to meet basic standards for characterization, emphasizing the importance of thorough validation .
A comprehensive validation protocol for PBLD antibodies should include:
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Positive and negative controls: Use cell lines or tissues with known PBLD expression levels as positive controls and PBLD-knockout or PBLD-negative samples as negative controls .
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Multi-method confirmation: Validate antibody specificity across different techniques (Western blot, IHC, IF) to ensure consistency between methods .
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Knockdown validation: Perform siRNA or CRISPR-based knockdown of PBLD and confirm reduced antibody signal proportional to knockdown efficiency .
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Peptide competition assay: Pre-incubate antibody with immunizing peptide/protein to demonstrate specific blocking of the signal .
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Molecular weight verification: Confirm that the detected band in Western blot corresponds to the expected molecular weight of PBLD (approximately 31.6 kDa) .
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Orthogonal validation: Compare results with alternative detection methods such as mass spectrometry or RNA expression data .
These validation steps are especially important for PBLD research, as multiple protein-domain containing proteins may share structural similarities that could lead to cross-reactivity.
What factors influence epitope accessibility when using PBLD antibodies?
Epitope accessibility can significantly impact experimental outcomes when using PBLD antibodies:
| Factor | Impact on Epitope Accessibility | Mitigation Strategy |
|---|---|---|
| Protein conformation | Native folding may mask epitopes | Use denaturing conditions for linear epitopes |
| Fixation method | Cross-linking can obscure epitopes | Optimize antigen retrieval protocols |
| Post-translational modifications | May alter epitope structure | Consider antibodies targeting unmodified regions |
| Protein-protein interactions | Partner proteins may block access | Use detergents or dissociation conditions |
| pH conditions | May affect antibody-antigen binding | Test pH optimization in binding buffers |
Recent research on pH-dependent antibodies demonstrates how binding affinity can vary significantly under different pH conditions . While not specifically documented for PBLD antibodies, this principle may be relevant for certain experimental designs, particularly when investigating PBLD in cellular compartments with varying pH.
For comprehensive protein complex studies, the approach described by researchers at Sanford Burnham Prebys could be adapted to PBLD research: using fusion proteins to stabilize protein complexes during immunization, enabling the generation of complex-specific antibodies .
How do monoclonal and polyclonal PBLD antibodies differ in research applications?
Understanding the fundamental differences between monoclonal and polyclonal PBLD antibodies is crucial for experimental design:
The choice between monoclonal and polyclonal antibodies should be guided by your experimental requirements. For highly specific detection of a particular PBLD epitope, monoclonal antibodies like clone 4F6 are preferable. For maximum sensitivity or detection of partially degraded proteins, polyclonal antibodies may be advantageous.
How can computational models improve antibody selection for PBLD research?
Recent advances in computational biology have created new opportunities for antibody selection and design:
Researchers have developed biophysics-informed models that can predict antibody binding modes and specificity profiles beyond those observed in experimental data . These computational approaches could be applied to PBLD antibody research in several ways:
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Epitope prediction: Computational tools can identify likely epitopes on the PBLD protein based on structural features and accessibility.
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Cross-reactivity assessment: Models can predict potential cross-reactivity with structurally similar proteins, helping researchers select more specific antibodies.
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Antibody engineering: For specialized applications, computational approaches can guide the design of antibodies with customized specificity profiles for PBLD.
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Binding mode analysis: Advanced models can distinguish between different binding modes, which is particularly important when studying protein-protein interactions involving PBLD.
According to research published in 2024, computational models trained on experimentally selected antibodies can associate distinct binding modes with potential ligands, enabling prediction and generation of specific variants . This approach could potentially be adapted to enhance PBLD antibody selection.
What are the optimal Western blotting conditions for PBLD antibodies?
Western blotting is one of the most common applications for PBLD antibodies. The following optimized protocol is based on validated methodologies:
For optimal results, perform a titration experiment to determine the ideal concentration of your specific PBLD antibody, as the optimal dilution can vary between antibody lots and sample types .
How should I troubleshoot non-specific binding with PBLD antibodies?
Non-specific binding is a common challenge when working with antibodies. Here's a systematic approach to troubleshooting:
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Identify the pattern of non-specific binding:
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Multiple unexpected bands in Western blot
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Background staining in IHC/IF
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Signal in negative control samples
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Optimization strategies based on root causes:
| Issue | Potential Cause | Solution |
|---|---|---|
| Multiple bands | Cross-reactivity | Use more stringent blocking (5% BSA instead of milk) |
| Protein degradation | Add protease inhibitors during sample preparation | |
| Antibody concentration too high | Increase antibody dilution (e.g., from 1:1000 to 1:2000) | |
| High background | Insufficient blocking | Extend blocking time or change blocking reagent |
| Inadequate washing | Increase number and duration of washes | |
| Secondary antibody issues | Include a secondary-only control | |
| False positives | Endogenous peroxidase activity | Add H₂O₂ quenching step for IHC |
| Fc receptor binding | Pre-block with species-matched normal serum |
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Advanced troubleshooting for persistent issues:
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Pre-adsorption: Incubate antibody with non-specific proteins or tissues
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Competition assay: Pre-incubate with immunizing peptide to confirm specificity
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Try alternative antibody clones targeting different PBLD epitopes
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Careful optimization and thorough controls are essential for distinguishing specific from non-specific signals, particularly when studying proteins like PBLD that may have structural similarities to other cellular proteins.
What is the optimal immunoprecipitation protocol for PBLD studies?
Immunoprecipitation (IP) is valuable for studying PBLD protein interactions and modifications. The following protocol is optimized for PBLD antibodies:
| Step | Protocol Details |
|---|---|
| Cell lysis | Use NP-40 or RIPA buffer with protease/phosphatase inhibitors |
| Pre-clearing | Incubate lysate with protein A/G beads for 1 hour at 4°C |
| Antibody binding | Add 2-5 μg PBLD antibody per 500 μg protein lysate |
| Incubate overnight at 4°C with gentle rotation | |
| Bead capture | Add pre-washed protein A/G beads, incubate 2-4 hours at 4°C |
| Washing | 4-5 washes with lysis buffer, final wash with PBS |
| Elution | SDS sample buffer at 95°C for 5 minutes |
| Controls | Include IgG isotype control (same species as PBLD antibody) |
| Input sample (pre-IP lysate) |
This protocol can be adapted for co-immunoprecipitation studies to investigate proteins that interact with PBLD. When selecting antibodies for IP, consider those that have been specifically validated for this application, as not all Western blot-validated antibodies work effectively for immunoprecipitation.
For studying protein complexes involving PBLD, research has shown that complex stability is critical . Consider crosslinking approaches or specialized buffers to maintain complex integrity during IP procedures.
How can I apply PBLD antibodies in phage display selections?
Phage display is a powerful technique for studying antibody-antigen interactions and can be applied to PBLD research. Based on methodologies described in recent literature:
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Library preparation:
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Selection strategy:
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Immobilize purified PBLD protein on a solid support
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Perform 2-3 rounds of selection with amplification steps between rounds
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Include pre-selection steps to deplete non-specific binders
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Analysis of selected phages:
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Use high-throughput sequencing to identify enriched antibody sequences
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Analyze the distribution of amino acids at key positions in the CDRs
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Express and purify promising candidates for further characterization
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This approach can be particularly valuable for identifying novel antibodies with unique binding properties to PBLD, or for studying the epitope landscape of PBLD to better understand its structure-function relationships.
Recent research has demonstrated how phage display selections against different ligand combinations can be used to train computational models for predicting antibody specificity . Such approaches could potentially be applied to develop highly specific PBLD antibodies.
What considerations apply to PBLD antibodies for studying protein complexes?
Recent research highlights challenges and solutions for studying protein complexes with antibodies:
Traditional antibody generation methods often fail with protein complexes because these complexes can be unstable during immunization . For PBLD complex studies, consider:
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Stabilization approach: Research published in March 2025 demonstrated that fusing protein complexes together adds stability during immunization, enabling antibody generation against complexes .
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Application to PBLD: If PBLD forms important complexes with other proteins, this fusion protein approach could generate antibodies specific to the complex rather than individual components.
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Quantitative detection: Such complex-specific antibodies allow direct measurement of complex formation on live cells, providing insights into regulatory mechanisms .
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Experimental design: When investigating if PBLD participates in protein complexes:
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Use mild lysis conditions to preserve native interactions
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Consider crosslinking to stabilize transient interactions
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Compare results using antibodies targeting different epitopes of PBLD
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Use proximity ligation assays to confirm direct interactions
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This advanced approach is particularly valuable if PBLD's function depends on interactions with partner proteins or if such interactions are altered in disease states.
How should PBLD antibodies be validated for publication-quality research?
The "antibody characterization crisis" has highlighted that many published studies use inadequately validated antibodies, undermining scientific reproducibility . For publication-quality PBLD research:
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Multi-level validation strategy:
| Validation Level | Required Evidence |
|---|---|
| Basic validation | Correct molecular weight detection on Western blot |
| Specific staining pattern in positive control samples | |
| Absence of signal in negative control samples | |
| Intermediate validation | Consistent results across multiple applications |
| Correlation with orthogonal methods (e.g., mRNA expression) | |
| Reproducibility across different experimental conditions | |
| Advanced validation | Signal reduction/elimination in knockout/knockdown models |
| Peptide competition assay showing specific blocking | |
| Mass spectrometry confirmation of immunoprecipitated proteins |
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Documentation requirements:
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Full antibody details: manufacturer, catalog number, lot number, clone
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Dilution used for each application
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Detailed methods including blocking, incubation times/temperatures
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All validation experiments performed
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Images of all controls
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Statement of antibody limitations:
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Explicitly state the conditions under which the antibody was validated
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Note any cross-reactivity observed
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Acknowledge any optimization limitations
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Complete and transparent reporting of antibody validation is essential for addressing the reproducibility challenges in antibody-based research .
