PLEKHJ1 Antibody

What is PLEKHJ1 and its biological significance?

PLEKHJ1 (Pleckstrin homology domain-containing family J member 1) is a human protein also known as PH domain-containing family J member 1 or Guanine nucleotide-releasing protein x (GNRPX) . The protein consists of 149 amino acids with a calculated molecular weight of approximately 18 kDa . It contains a pleckstrin homology domain, which typically binds phosphatidylinositol lipids and proteins, suggesting its potential role in cell signaling pathways. PLEKHJ1 is primarily studied in cell signaling research and may be involved in cell trafficking mechanisms . The protein has been assigned the UniProt ID Q9NW61 and NCBI Gene ID 55111 .

Understanding this protein's function requires experimentation with specific antibodies in various contexts, including cell signaling cascades, protein-protein interactions, and potential disease associations. Research typically involves protein localization studies, co-immunoprecipitation experiments, and functional assays to elucidate its biological roles.

What types of PLEKHJ1 antibodies are available for research?

The available PLEKHJ1 antibodies for research include both polyclonal and monoclonal variants with different conjugations and host species:

Each antibody type offers specific advantages. Polyclonal antibodies recognize multiple epitopes, providing robust signal detection but potentially lower specificity. Monoclonal antibodies target single epitopes, offering high specificity but potentially lower sensitivity. Selection should be based on experimental requirements, with monoclonals preferred for reproducible quantitative assays and polyclonals for applications requiring enhanced signal detection .

How should PLEKHJ1 antibodies be stored to maintain optimal activity?

Proper storage of PLEKHJ1 antibodies is critical for maintaining antibody integrity and experimental reproducibility. The polyclonal biotin-conjugated antibody should be initially shipped at 4°C, then upon delivery, aliquoted and stored at -20°C for short-term or -80°C for long-term storage . It's essential to avoid repeated freeze-thaw cycles, which can denature the antibody and reduce effectiveness.

The monoclonal unconjugated antibody (60453-1-PBS) should be stored at -80°C in its PBS-only buffer formulation . When preparing working aliquots, researchers should:

• Thaw antibodies on ice

• Create single-use aliquots (typically 5-10 μL) in sterile microcentrifuge tubes

• Return unused portions to -80°C immediately

• Avoid repeated freeze-thaw cycles by using fresh aliquots for each experiment

• Monitor storage conditions with temperature logs to ensure consistency

Failure to follow appropriate storage protocols may result in antibody degradation, leading to reduced binding efficiency and inconsistent experimental results.

What are the validated applications for PLEKHJ1 antibodies?

PLEKHJ1 antibodies have been validated for specific applications based on their format and characteristics. The polyclonal biotin-conjugated antibody (A67409-050) has been validated primarily for ELISA applications . The monoclonal antibody pair (60453-1-PBS capture and 60453-2-PBS detection) has been specifically validated for cytometric bead array applications .

Beyond these validated applications, researchers can optimize these antibodies for additional techniques through careful validation:

• Western Blotting: Optimization would include titration experiments (typically starting at 1:500 to 1:2000 dilutions), testing various blocking agents, and confirming specificity with appropriate controls.

• Immunoprecipitation: The monoclonal antibody may be suitable for immunoprecipitation after conjugation to appropriate beads, requiring optimization of antibody-to-bead ratios and washing conditions.

• Immunohistochemistry/Immunofluorescence: Though not explicitly validated, the unconjugated monoclonal antibody could potentially be optimized for these applications through antigen retrieval method testing and titration.

• Flow Cytometry: The biotin-conjugated antibody may be suitable for flow cytometry when paired with appropriate streptavidin-fluorophore conjugates.

When adapting PLEKHJ1 antibodies for non-validated applications, researchers should conduct preliminary validation experiments including positive and negative controls, concentration optimization, and specificity tests.

How can I optimize ELISA protocols specifically for PLEKHJ1 detection?

Optimizing ELISA protocols for PLEKHJ1 detection requires systematic adjustment of multiple parameters:

• Antibody Selection and Configuration:

  • For sandwich ELISA: Use the matched pair (60453-1-PBS as capture and 60453-2-PBS as detection antibody)

  • For direct ELISA: The biotin-conjugated polyclonal antibody (A67409-050) can be employed

• Coating Concentration Optimization:

  • Perform a titration experiment with capture antibody concentrations ranging from 0.5-10 μg/mL

  • Typical optimal concentration is 1-2 μg/mL in carbonate/bicarbonate buffer (pH 9.6)

• Blocking Optimization:

  • Test multiple blocking agents (1-5% BSA, 1-5% non-fat milk, commercial blocking buffers)

  • Include 0.05% Tween-20 to reduce background

• Sample and Detection Antibody Parameters:

  • Prepare standard curves using recombinant PLEKHJ1 protein (1-149AA) as reference

  • For the detection step with biotin-conjugated antibody, optimize concentration (typically 0.1-0.5 μg/mL)

  • Incubation time and temperature affect sensitivity (1-2 hours at room temperature or overnight at 4°C)

• Signal Development and Detection:

  • For biotin-conjugated antibodies, use streptavidin-HRP at optimized concentration (typically 1:5000-1:20000)

  • Substrate selection (TMB, ABTS, or OPD) affects sensitivity and dynamic range

  • Monitor kinetics of color development to determine optimal reading times

The substrate/buffer composition (0.03% Proclin 300, 50% Glycerol, 0.01M PBS, pH 7.4) of the polyclonal antibody should be considered when designing experiments to avoid buffer incompatibilities .

What are the considerations for using PLEKHJ1 antibodies in multiplexed assays?

Multiplexed assays allow simultaneous detection of multiple targets, but require careful consideration when incorporating PLEKHJ1 antibodies:

• Antibody Compatibility:

  • The unconjugated monoclonal antibody (60453-1-PBS) is specifically designed for multiplexed applications including cytometric bead arrays and multiplex imaging

  • Being provided in PBS-only format (BSA and azide-free) at 1 mg/mL, it's ready for conjugation to various detection systems

• Cross-Reactivity Assessment:

  • Perform pre-testing to ensure no cross-reactivity with other targets in the multiplex panel

  • Design experiments with appropriate single-analyte controls alongside multiplexed samples

  • Conduct spike-and-recovery experiments with known concentrations of purified PLEKHJ1

• Signal Separation Strategies:

  • For fluorescence-based multiplexing, select fluorophores with minimal spectral overlap

  • In bead-based assays, use distinct bead populations with appropriate separation

  • In mass cytometry applications, conjugate antibodies to distinct metal isotopes

• Optimization Protocols:

  • Titrate antibody concentration independently and in multiplexed format

  • Adjust incubation times to balance signal development across all targets

  • Establish independent standard curves for PLEKHJ1 in both single-target and multiplexed formats

• Data Analysis Considerations:

  • Apply appropriate compensation matrices for fluorescence spillover

  • Use specific software tools designed for multiplex data interpretation

  • Consider statistical approaches that account for multiple testing when analyzing results

The monoclonal format of 60453-1-PBS offers advantages in multiplexed settings due to its high specificity and reproducibility compared to polyclonal alternatives .

How can PLEKHJ1 antibodies be utilized in cell signaling pathway investigations?

PLEKHJ1 is categorized within the cell signaling research area, indicating its potential involvement in signal transduction pathways . To investigate its role in these pathways:

• Pathway Mapping Approaches:

  • Use phospho-specific antibodies alongside PLEKHJ1 antibodies to track activation states

  • Implement co-immunoprecipitation with PLEKHJ1 antibodies followed by mass spectrometry to identify binding partners

  • Employ proximity ligation assays to visualize protein-protein interactions in situ

• Perturbation Experiments:

  • Combine antibody-based detection with genetic manipulation (siRNA knockdown, CRISPR-Cas9 editing)

  • Use specific pathway inhibitors to determine where PLEKHJ1 functions within signaling cascades

  • Apply stimulus-response experiments with time-course antibody detection

• Subcellular Localization Studies:

  • Perform fractionation experiments with subsequent Western blotting using PLEKHJ1 antibodies

  • Conduct co-localization studies using confocal microscopy with PLEKHJ1 antibodies and organelle markers

  • Track translocation events following cellular stimulation

• Signal Quantification Methods:

  • Implement quantitative Western blotting with appropriate loading controls

  • Use high-content imaging to quantify spatial and temporal changes in PLEKHJ1 expression

  • Apply phospho-flow cytometry techniques if PLEKHJ1 phosphorylation states are relevant

These approaches can elucidate PLEKHJ1's position within signaling networks, potentially revealing new therapeutic targets or biomarkers related to cell signaling dysregulation.

What are the best practices for validating PLEKHJ1 antibody specificity?

Rigorous validation of antibody specificity is crucial for generating reliable research data. For PLEKHJ1 antibodies, implement the following validation strategies:

• Genetic Validation Approaches:

  • Test antibody reactivity in PLEKHJ1 knockout/knockdown models

  • Perform antibody testing on cells overexpressing tagged PLEKHJ1

  • Utilize siRNA knockdown with decreasing protein expression confirmed by the antibody

• Peptide Competition Assays:

  • Pre-incubate antibody with excess immunizing peptide or recombinant PLEKHJ1 protein

  • Compare signal between blocked and unblocked antibody samples

  • For the polyclonal antibody, use the recombinant human PLEKHJ1 protein (1-149AA) that served as the immunogen

• Orthogonal Detection Methods:

  • Compare antibody-based detection with mass spectrometry results

  • Validate findings using multiple antibodies recognizing different epitopes

  • Compare the polyclonal (A67409-050) and monoclonal (60453-1-PBS) results for concordance

• Cross-Reactivity Assessment:

  • Test reactivity against closely related proteins in the pleckstrin homology domain family

  • Evaluate species cross-reactivity (the provided antibodies are indicated for human reactivity)

  • Test in tissues known to have varying expression levels of PLEKHJ1

• Application-Specific Controls:

  • Include isotype controls (mouse IgG1 for the monoclonal antibody)

  • Use secondary-only controls to assess background

  • Include positive control samples with known PLEKHJ1 expression

Comprehensive validation experiments should be performed for each new lot of antibody and for each experimental application to ensure reproducibility and reliability of results.

How can PLEKHJ1 antibodies be integrated into advanced imaging workflows?

Integrating PLEKHJ1 antibodies into advanced imaging workflows requires careful optimization and specialized techniques:

• Super-Resolution Microscopy Applications:

  • For STORM/PALM: Conjugate the unconjugated monoclonal antibody (60453-1-PBS) to photoswitchable fluorophores

  • For STED microscopy: Use fluorophores with appropriate depletion properties

  • Optimize labeling density to achieve sufficient spatial resolution without overcrowding

• Multiplexed Imaging Strategies:

  • Implement cyclic immunofluorescence with antibody stripping and re-probing

  • Use spectral unmixing approaches when employing multiple fluorophores

  • Apply tissue clearing techniques for three-dimensional imaging in thick specimens

• Live-Cell Imaging Considerations:

  • Develop cell-permeable PLEKHJ1 antibody fragments for live-cell applications

  • Consider antibody-fluorescent protein fusions for dynamic tracking

  • Optimize antibody concentration to minimize perturbation of cellular functions

• Correlative Light and Electron Microscopy (CLEM):

  • Conjugate PLEKHJ1 antibodies with electron-dense particles (e.g., gold nanoparticles)

  • Develop protocols for sample preparation that preserve both antigenicity and ultrastructure

  • Establish registration methods to correlate fluorescence and electron microscopy images

• Image Analysis Workflows:

  • Implement segmentation algorithms to quantify PLEKHJ1 distribution patterns

  • Develop co-localization analysis pipelines for multi-channel imaging

  • Utilize machine learning approaches for pattern recognition in complex tissues

The unconjugated format of the monoclonal antibody (60453-1-PBS) makes it particularly suitable for custom conjugation to various fluorophores or nanoparticles for specialized imaging applications .

How should I address inconsistent results with PLEKHJ1 antibodies?

Inconsistent results when using PLEKHJ1 antibodies can stem from multiple sources. A systematic troubleshooting approach includes:

• Antibody-Related Factors:

  • Verify antibody integrity by testing aliquots stored under different conditions

  • Check for lot-to-lot variability by requesting Certificate of Analysis data

  • Consider antibody degradation if using beyond recommended storage time

  • For the biotin-conjugated antibody, test biotin detection system separately

• Sample Preparation Issues:

  • Optimize protein extraction methods (different lysis buffers may affect epitope accessibility)

  • For fixed samples, evaluate different fixation protocols (paraformaldehyde vs. methanol)

  • Standardize sample handling time to prevent protein degradation

  • Include protease/phosphatase inhibitors in lysis buffers

• Protocol Optimization:

  • Systematically vary antibody concentration, incubation time, and temperature

  • For ELISA applications, optimize blocking agents to reduce background

  • Adjust buffer conditions (salt concentration, pH) to improve signal-to-noise ratio

  • Modify antigen retrieval methods for fixed tissue samples

• Control Implementation:

  • Include positive controls with known PLEKHJ1 expression

  • Run parallel experiments with different PLEKHJ1 antibodies (polyclonal vs. monoclonal)

  • Implement loading controls for normalization in Western blots

  • Use isotype controls to assess non-specific binding

• Technical Variables:

  • Standardize equipment settings across experiments

  • Calibrate instruments regularly

  • Maintain detailed records of experimental conditions

  • Consider environmental factors (temperature, humidity) that may affect results

Creating a standardized operating procedure (SOP) with clearly defined parameters and quality control checkpoints can significantly improve reproducibility when working with PLEKHJ1 antibodies.

How can I distinguish between specific and non-specific binding of PLEKHJ1 antibodies?

Distinguishing specific from non-specific binding is crucial for accurate interpretation of PLEKHJ1 antibody results:

• Control-Based Approaches:

  • Compare signal patterns between wild-type and PLEKHJ1-knockout/knockdown samples

  • Implement peptide competition assays using the recombinant PLEKHJ1 protein (1-149AA)

  • Use isotype controls (mouse IgG1 for monoclonal antibody) to assess Fc-mediated binding

  • Include secondary antibody-only controls to evaluate background

• Signal Evaluation Methods:

  • Assess signal pattern consistency with predicted cellular localization

  • Compare molecular weight in Western blots with the expected 18 kDa for PLEKHJ1

  • Evaluate dose-response relationships in antigen titration experiments

  • Analyze sigmoidal binding curves characteristic of specific antibody-antigen interactions

• Cross-Validation Strategies:

  • Compare results from multiple antibodies targeting different PLEKHJ1 epitopes

  • Validate antibody-based findings with non-antibody methods (e.g., mass spectrometry)

  • Test antibody in multiple applications to confirm consistent target recognition

  • Compare results from both polyclonal and monoclonal antibodies

• Technical Optimization:

  • Increase stringency of washing steps to reduce non-specific binding

  • Optimize blocking conditions (test different blocking agents and concentrations)

  • Adjust antibody concentration to improve signal-to-noise ratio

  • Consider detergent types and concentrations in buffers

• Data Analysis Approaches:

  • Implement quantitative thresholding based on control samples

  • Use statistical methods to distinguish signal from background

  • Apply computational approaches to identify true positive signals

These approaches collectively provide a framework for confidence in the specificity of observed PLEKHJ1 signals across different experimental contexts.

What are the limitations of current PLEKHJ1 antibodies in research applications?

Understanding the limitations of current PLEKHJ1 antibodies is essential for proper experimental design and data interpretation:

• Epitope Accessibility Constraints:

  • The polyclonal antibody recognizes multiple epitopes within the recombinant PLEKHJ1 protein (1-149AA), but may have limited access to certain conformations

  • Post-translational modifications may mask epitopes

  • Protein-protein interactions may obscure antibody binding sites

  • Fixation methods can alter epitope structure and accessibility

• Cross-Reactivity Considerations:

  • The available antibodies have been primarily tested for human PLEKHJ1 reactivity

  • Cross-reactivity with other species is not fully characterized

  • Potential cross-reactivity with other pleckstrin homology domain-containing proteins

  • Background in certain tissue types may limit sensitivity

• Application-Specific Limitations:

  • The biotin-conjugated antibody has been primarily validated for ELISA applications

  • The monoclonal pair is optimized for cytometric bead arrays

  • Performance in other applications requires additional validation

  • Sensitivity limitations in detecting low abundance PLEKHJ1

• Technical Challenges:

  • The 18 kDa size of PLEKHJ1 may be challenging to resolve on standard Western blots

  • Quantitative applications may be limited by narrow dynamic ranges

  • Batch-to-batch variability, particularly with polyclonal antibodies

  • Limited data on epitope mapping affects ability to interpret negative results

• Functional Limitations:

  • Current antibodies may detect protein presence but not functional state

  • Limited availability of phospho-specific or other modification-specific antibodies

  • Potential interference with protein function in certain applications

  • Inability to distinguish between splice variants

Researchers should consider these limitations when designing experiments and interpreting results, potentially implementing complementary approaches to address specific limitations of antibody-based detection.

How might new antibody technologies improve PLEKHJ1 research?

Emerging antibody technologies offer significant potential for advancing PLEKHJ1 research:

• Next-Generation Antibody Development:

  • Recombinant antibody production could improve lot-to-lot consistency compared to current polyclonal and hybridoma-derived antibodies

  • Single-domain antibodies (nanobodies) may access epitopes unreachable by conventional antibodies

  • Phage display libraries could generate highly specific PLEKHJ1 binders with defined properties

  • Antibody engineering could create bispecific formats for co-detection of PLEKHJ1 with interaction partners

• Advanced Conjugation Strategies:

  • Site-specific conjugation techniques could improve the current biotin conjugation approach

  • Conjugation to DNA barcodes would enable highly multiplexed detection in CODEX systems

  • Photocaged antibodies could allow spatiotemporal control of PLEKHJ1 detection

  • Click chemistry-compatible antibodies would enable modular functionalization

• Improved Screening Methodologies:

  • Next-generation sequencing approaches described in search result could be applied to developing more diverse PLEKHJ1 antibody pools

  • The Golden Gate-based dual-expression vector system could accelerate PLEKHJ1 antibody development

  • Integration with computational antibody design could optimize binding properties

  • High-throughput epitope mapping would create epitope-specific antibody panels

• Innovative Detection Systems:

  • Proximity-based detection methods (PLA, FRET) could reveal PLEKHJ1 interaction networks

  • Label-free detection technologies might overcome limitations of current conjugated systems

  • Single-molecule detection approaches could improve sensitivity for low-abundance PLEKHJ1

  • In vivo imaging probes based on PLEKHJ1 antibodies could track dynamic processes

• Functional Antibody Development:

  • Conformation-specific antibodies could distinguish active/inactive PLEKHJ1 states

  • Intrabodies engineered for intracellular expression could track PLEKHJ1 in living cells

  • Antibody-drug conjugates might target cells with aberrant PLEKHJ1 expression

  • Allosteric modulatory antibodies could probe PLEKHJ1 function

The methodologies described for antibody development in search result , including NGS technology and Golden Gate Cloning for dual-expression vectors, represent promising approaches that could be adapted specifically for PLEKHJ1 antibody development.

What unexplored applications might benefit from PLEKHJ1 antibody research?

Several unexplored research areas could benefit from improved PLEKHJ1 antibody tools:

• Biomarker Development:

  • Investigation of PLEKHJ1 as a potential diagnostic or prognostic biomarker

  • Development of clinical-grade assays using the available matched antibody pair

  • Exploration of PLEKHJ1 levels in various disease states

  • Correlation of PLEKHJ1 expression with disease progression or treatment response

• Cell Trafficking Research:

  • Given the keyword association with "Cell Trafficking" , investigation of PLEKHJ1's role in vesicular transport

  • Studies on PLEKHJ1 involvement in endocytosis/exocytosis pathways

  • Exploration of potential roles in protein secretion or membrane recycling

  • Investigation of PLEKHJ1 in polarized cell trafficking

• Structural Biology Applications:

  • Use of antibodies as crystallization chaperones for PLEKHJ1 structural studies

  • Epitope mapping to identify functional domains within the 149 amino acid protein

  • Antibody-based studies of PLEKHJ1 conformational changes

  • Investigation of the pleckstrin homology domain's binding partners

• Developmental Biology:

  • Tracking PLEKHJ1 expression patterns during development

  • Investigation of PLEKHJ1 in cell differentiation processes

  • Studies on potential roles in tissue morphogenesis

  • Exploration of PLEKHJ1 in stem cell biology

• Therapeutic Applications:

  • Evaluation of PLEKHJ1 as a potential therapeutic target

  • Development of function-modulating antibodies

  • Investigation of PLEKHJ1 in drug resistance mechanisms

  • Exploration of PLEKHJ1's role in disease pathways

The matched antibody pair format (60453-1-PBS capture and 60453-2-PBS detection) is particularly well-suited for quantitative applications that could support biomarker research .

How can researchers contribute to improving PLEKHJ1 antibody validation standards?

Researchers can significantly contribute to improving validation standards for PLEKHJ1 antibodies through several approaches:

• Enhanced Validation Reporting:

  • Document comprehensive validation experiments for each application

  • Share detailed protocols including optimization parameters

  • Report negative results alongside positive findings

  • Create and contribute to antibody validation databases specific to PLEKHJ1

• Multi-Method Validation Approaches:

  • Implement orthogonal methods to confirm antibody specificity

  • Conduct genetic validation using CRISPR-Cas9 PLEKHJ1 knockout models

  • Perform cross-platform validation (e.g., comparing Western blot with mass spectrometry)

  • Validate across multiple sample types and preparation methods

• Collaborative Validation Initiatives:

  • Participate in multi-laboratory validation studies

  • Contribute to antibody validation consortia

  • Share reference samples with established PLEKHJ1 expression profiles

  • Develop community standards for PLEKHJ1 antibody characterization

• Advanced Validation Technologies:

  • Apply emerging technologies like CRISPR-Cas9 epitope tagging

  • Implement proteogenomic approaches to correlate antibody binding with expression data

  • Utilize mass spectrometry immunoprecipitation to confirm target specificity

  • Develop quantitative metrics for validation rather than binary assessments

• Open Science Practices:

  • Publish detailed antibody characterization data through repositories

  • Share raw validation data alongside processed results

  • Contribute to community resources like Antibodypedia with PLEKHJ1-specific information

  • Develop open standards for PLEKHJ1 antibody validation