PLEK Antibody

What is PLEK and why is it significant in hematopoietic research?

PLEK (pleckstrin, also known as p47) was originally identified as the major PKC substrate in platelets and is expressed in all cells of the hematopoietic system. Its significance derives from its structural features, particularly the two pleckstrin homology (PH) domains at its N and C termini, which enable interactions with various protein and lipid ligands. This allows PLEK to function as an intracellular adaptor/targeting protein. In unstimulated cells, PLEK is predominantly found in the cytosol, suggesting its role in cellular signaling pathways .

What are the key differences between polyclonal and monoclonal PLEK antibodies for research applications?

Polyclonal PLEK antibodies (such as 12506-1-AP) and monoclonal PLEK antibodies (such as 66431-1-Ig) have distinct characteristics that affect their research utility:

Selecting between these antibody types depends on the experimental goals. Polyclonal antibodies offer greater signal amplification and robustness against sample preparation variations, while monoclonal antibodies provide higher specificity and consistency between batches .

What are the optimal buffer conditions for antigen retrieval when using PLEK antibodies in immunohistochemistry?

Based on validated protocols, two buffer systems have been proven effective for antigen retrieval when using PLEK antibodies in immunohistochemistry:

• Primary recommendation: TE buffer at pH 9.0

• Alternative method: Citrate buffer at pH 6.0

The choice between these buffers may depend on the specific tissue type and fixation method. For example, positive IHC signals have been detected in human lung cancer tissue using the 12506-1-AP antibody and human colon cancer tissue using the 66431-1-Ig antibody. Researchers should empirically optimize the antigen retrieval conditions for their specific tissue samples, particularly when working with tissues that differ from the validated samples .

How should dilution optimization be approached for new experimental systems using PLEK antibodies?

When establishing PLEK antibody protocols in new experimental systems, a systematic dilution optimization approach is recommended:

• Start with the manufacturer's recommended dilution range:

  • For Western blot: 1:1000-1:4000 (polyclonal) or 1:2000-1:20000 (monoclonal)

  • For IHC: 1:20-1:200 (polyclonal) or 1:50-1:500 (monoclonal)

  • For IF-P: 1:200-1:800 (monoclonal)

• Perform a preliminary experiment using 3-4 dilutions spanning the recommended range

• Evaluate signal-to-noise ratio, background staining, and specific target detection

• Refine the dilution range based on initial results and perform a secondary validation

• Include appropriate positive controls (such as U-937 cells, rat spleen tissue for WB, or human lung/colon cancer tissue for IHC)

This titration approach is essential as optimal dilutions are sample-dependent and may vary based on protein expression levels, tissue type, and detection methods .

What storage conditions maximize PLEK antibody stability and shelf life?

To maintain PLEK antibody functionality and extend shelf life, the following storage protocols should be implemented:

• Store antibodies at -20°C in their original buffer (PBS with 0.02% sodium azide and 50% glycerol, pH 7.3)

• For antibodies in the 20μl size, note that they contain 0.1% BSA as a stabilizer

• According to manufacturer specifications, aliquoting is unnecessary for -20°C storage, which simplifies laboratory workflow

• Antibodies remain stable for one year after shipment when stored under these conditions

• For working solutions, minimize freeze-thaw cycles by preparing single-use aliquots if the antibody will be used repeatedly

These storage guidelines ensure consistent antibody performance across experimental timeframes .

How can PLEK antibodies be incorporated into multiparameter flow cytometry for hematopoietic cell analysis?

Given PLEK's expression in all hematopoietic cells, its antibodies can serve as valuable markers in multiparameter flow cytometry. A methodological approach includes:

• Sample preparation: Isolate peripheral blood mononuclear cells (PBMCs) or bone marrow cells using density gradient centrifugation

• Surface marker staining: First label cells with fluorochrome-conjugated antibodies against lineage-specific surface markers (CD3, CD19, CD14, etc.)

• Fixation and permeabilization: Use a commercial kit designed for intracellular antigens

• PLEK staining: Apply anti-PLEK antibody (optimally diluted) followed by appropriate secondary antibody if using unconjugated primary

• Analysis gating strategy:

  • First gate on viable cells

  • Identify cell populations based on lineage markers

  • Quantify PLEK expression within each lineage

  • Compare PLEK expression levels between different hematopoietic populations

This approach allows researchers to correlate PLEK expression with specific hematopoietic cell types and activation states .

What strategies can resolve discrepancies in PLEK detection between different antibody clones?

When facing inconsistent results between different PLEK antibody clones, implement this systematic troubleshooting approach:

• Epitope mapping: Determine which region of PLEK each antibody recognizes (N-terminal PH domain, C-terminal PH domain, or intervening sequences)

• Cross-validation: Test multiple antibodies targeting different epitopes on the same samples

• Validation controls:

  • Positive controls: Use cell lines with confirmed PLEK expression (U-937, THP-1, HL-60)

  • Negative controls: Consider PLEK knockdown or knockout samples, which have been validated in published studies

  • Tissue controls: Use rat spleen, pig spleen, or mouse spleen tissues as positive controls

• Methodological validation: If discrepancies persist, validate results with complementary techniques (e.g., mass spectrometry) to confirm protein identity

• Documentation: Record the exact antibody clone, lot number, and experimental conditions to facilitate reproducibility and troubleshooting

This approach can help distinguish between true biological variations and technical artifacts in PLEK detection .

How does phosphorylation status affect PLEK detection and what methods can assess PLEK phosphorylation state?

As the major substrate of PKC in platelets, PLEK's phosphorylation state is critical to its function. This creates important considerations for antibody-based detection:

• Band shift analysis: Phosphorylated PLEK typically migrates more slowly in SDS-PAGE, contributing to the observed 40-47 kDa range in Western blots

• Phosphorylation-specific detection methods:

  • Use phosphorylation-specific antibodies (if available)

  • Perform lambda phosphatase treatment on parallel samples to confirm phosphorylation-dependent mobility shifts

  • Use Phos-tag™ acrylamide gels to enhance separation of phosphorylated species

• Stimulation experiments: Compare PLEK detection in resting cells versus cells stimulated with PKC activators (e.g., PMA) to assess dynamic phosphorylation

• Quantification considerations: When quantifying PLEK by Western blot, consider whether to measure a specific band or integrate across the entire 40-47 kDa range depending on the research question

Understanding these phosphorylation-related aspects is crucial for accurate interpretation of PLEK antibody results, particularly in signaling studies .

How can deep learning approaches enhance PLEK antibody design and validation?

Recent advances in computational methods offer promising approaches for PLEK antibody research:

Deep learning-based antibody design can generate in-silico sequences with optimized properties. This approach combines:

• Sequence and structure-based deep learning to predict mutation effects on antibody properties

• Multi-objective linear programming with diversity constraints to yield high-performing antibody libraries

• Validation through expression testing in mammalian cells, which has shown that algorithmically-designed antibodies express well and can be purified in sufficient quantities

For PLEK antibody validation, researchers can apply similar computational approaches to analyze antibody binding characteristics and optimize experimental protocols. This computational-experimental hybrid approach represents the cutting edge of antibody research methodology .

What considerations are important when selecting matched antibody pairs for PLEK detection in sandwich ELISA or other dual-recognition assays?

When establishing a sandwich ELISA or other dual-recognition assay for PLEK, consider these methodological factors:

• Epitope compatibility: Select capture and detection antibodies targeting non-overlapping epitopes to enable simultaneous binding

• Optimized antibody pair sets: Commercial matched pairs (such as the Abnova H00005341-AP51) typically include:

  • Capture antibody: Mouse monoclonal anti-PLEK

  • Detection antibody: Rabbit polyclonal anti-PLEK

• Cross-reactivity testing: Validate that the antibody pair does not produce false positives with related proteins containing PH domains

• Sample matrix optimization: Determine if sample diluent components interfere with antibody binding or create background

• Standard curve development: Use recombinant PLEK protein to establish a quantitative relationship between signal and concentration

• Assay validation:

  • Determine lower limit of detection and quantification

  • Assess intra- and inter-assay variability

  • Confirm linearity in the working range

  • Test spike recovery to assess matrix effects

This systematic approach ensures reliable quantification of PLEK in complex biological samples .

What specialized methods can distinguish between the cytosolic and membrane-translocated forms of PLEK protein?

Since PLEK translocates from cytosol to membrane upon cellular activation, specialized methods are needed to study this dynamic process:

• Subcellular fractionation approach:

  • Separate cytosolic and membrane fractions through ultracentrifugation

  • Confirm fraction purity with markers (e.g., Na+/K+-ATPase for membrane, GAPDH for cytosol)

  • Quantify PLEK in each fraction by Western blot

• Live-cell imaging methods:

  • Generate PLEK-GFP fusion constructs for real-time translocation monitoring

  • Validate that the antibody recognizes the fusion protein

  • Use confocal microscopy to track PLEK translocation during cell stimulation

• Proximity ligation assay (PLA):

  • Use PLEK antibody in combination with membrane marker antibodies

  • PLA signal indicates when PLEK is in close proximity to membrane components

• Immunofluorescence analysis:

  • Use the 66431-1-Ig antibody validated for IF-P (1:200-1:800 dilution)

  • Co-stain with membrane markers

  • Perform ratio imaging to quantify cytosolic versus membrane-associated PLEK

These approaches provide complementary data on PLEK translocation dynamics in response to cellular stimulation .

What are the most common causes of non-specific binding with PLEK antibodies and how can they be addressed?

When encountering non-specific binding with PLEK antibodies, implement this systematic troubleshooting approach:

• Common causes and solutions:

• Validation strategies:

  • Include a PLEK knockout/knockdown control

  • Perform peptide competition assay

  • Compare results from multiple antibodies targeting different epitopes

• Sample preparation considerations:

  • For WB: Ensure complete protein denaturation

  • For IHC/IF: Optimize fixation time and antigen retrieval conditions

These approaches minimize non-specific signals and increase confidence in PLEK detection specificity .

How should researchers validate PLEK antibody specificity in their experimental system?

A comprehensive validation strategy for PLEK antibodies includes:

• Literature-based validation:

  • Review published applications of the antibody

  • Assess citations for the specific antibody clone

  • Examine validation data from antibody providers

• Technical validation sequence:

  • Western blot analysis using positive control samples (U-937, THP-1, HL-60 cells or rat/pig spleen tissue)

  • Molecular weight confirmation (40-47 kDa range)

  • Testing in multiple applications (WB, IHC, IF) to ensure consistent results

• Biological validation:

  • Include tissues/cells known to express PLEK (hematopoietic cells)

  • Include non-hematopoietic controls (expected to be negative)

  • Test expression in response to known PLEK regulators

• Advanced validation approaches:

  • Genetic knockdown/knockout verification

  • Mass spectrometry confirmation of immunoprecipitated protein

  • Immunodepletion experiments

Researchers should select validation methods appropriate to their experimental system and document validation results to support publication-quality data .

What emerging applications represent the future of PLEK antibody utilization in hematopoietic research?

PLEK antibody research is evolving toward several promising directions:

• Single-cell analysis: Integration of PLEK antibodies into CyTOF and single-cell proteomics workflows to analyze PLEK expression heterogeneity within hematopoietic cell populations

• Spatial biology: Incorporation of PLEK antibodies into multiplexed tissue imaging platforms (e.g., CODEX, Imaging Mass Cytometry) to map PLEK expression within tissue microenvironments

• Synthetic biology approaches: Development of PLEK-based biosensors to monitor real-time PKC activity in living cells

• Therapeutic potential: Investigation of PLEK as a target in hematological disorders where platelet function is dysregulated

• Computational integration: Application of deep learning approaches to predict PLEK antibody binding characteristics and optimize experimental protocols

These emerging applications highlight the continued value of well-validated PLEK antibodies in advancing our understanding of hematopoietic cell biology and potential therapeutic interventions .

For longitudinal clinical studies involving PLEK antibodies, researchers should implement these quality control measures:

• Batch consistency strategies:

  • Purchase sufficient antibody from a single lot for the entire study duration

  • If multiple lots must be used, perform cross-lot validation

  • Include consistent positive controls in each experiment

• Sample processing standardization:

  • Establish fixed protocols for sample collection, processing, and storage

  • Document processing time and temperature

  • Use automated systems where possible to minimize operator variability

• Quantification approaches:

  • Develop validated quantitative assays (e.g., sandwich ELISA)

  • Include standard curves in each assay run

  • Use digital image analysis for IHC quantification to reduce subjective interpretation

• Quality control monitoring:

  • Track antibody performance over time with control samples

  • Monitor inter-operator and inter-site variability

  • Establish acceptance criteria for assay validity

• Data integration considerations:

  • Develop standardized data collection forms

  • Establish clear procedures for handling outliers

  • Plan appropriate statistical approaches for longitudinal data analysis