PXN Antibody, Biotin conjugated

What are the optimal storage conditions for biotin-conjugated PXN antibodies?

Biotin-conjugated PXN antibodies should be stored at -20°C for long-term storage (up to one year from date of receipt). For frequent use over shorter periods (up to one month), storage at 4°C is acceptable. It's crucial to avoid repeated freeze-thaw cycles as these can degrade antibody quality and performance. When preparing small working aliquots for freezing at -20°C, addition of cryoprotectants such as glycerol is recommended to maintain antibody integrity .

How do I select the appropriate biotin-conjugated PXN antibody for my experiment?

Selection should be based on several critical factors:

For maximum detection sensitivity when using biotin conjugates, antibodies with spacers (like biotin-SP) provide better accessibility to streptavidin binding sites .

Can I conjugate commercial PXN antibodies with biotin myself, and what protocol should I follow?

Yes, commercial PXN antibodies can be conjugated with biotin in laboratory settings. The procedure involves:

• Buffer exchange to remove interfering substances (sodium azide, BSA) using dialysis or desalting columns

• Reaction with biotin-NHS ester at optimal pH (typically 8.0-8.5)

• Purification to remove unreacted biotin

• Storage in buffer containing cryoprotectant

Limitations to consider:

• Carrier proteins like BSA must be removed before conjugation as they compete with the antibody for biotin binding sites

• Sodium azide should be eliminated as it can interfere with certain conjugation chemistries

• Post-conjugation, addition of stabilizers like trehalose or glycerol is recommended for storage at -20°C

What are the optimal dilutions for biotin-conjugated PXN antibodies in different applications?

These recommendations provide starting points; optimal concentrations should be determined experimentally for each specific application and sample type .

How do I design experiments to study PXN phosphorylation dynamics using biotin-conjugated antibodies?

To effectively study PXN phosphorylation dynamics:

• Experimental setup:

  • Use paired antibodies: total PXN (A01033-1 or A01033-2) and phospho-specific (A01033Y118 for pY118)

  • Include appropriate controls (untreated/unstimulated cells, phosphatase treatment)

  • Consider time-course experiments to capture temporal dynamics

• Detection system:

  • For biotin-conjugated antibodies, use streptavidin conjugated to appropriate reporter (HRP, fluorophore)

  • For imaging applications, streptavidin-conjugated fluorophores allow visualization of spatial distribution of phosphorylated PXN

  • For quantitative analysis, flow cytometry with biotin-conjugated PXN antibodies plus streptavidin-fluorophore provides statistical power

• Data analysis:

  • Calculate phospho-PXN:total PXN ratio to normalize for expression level variations

  • Consider subcellular localization when interpreting results, as phosphorylation may affect protein distribution

What controls should be included when using biotin-conjugated PXN antibodies in immunofluorescence?

A robust immunofluorescence experiment using biotin-conjugated PXN antibodies should include:

For multicolor experiments, single-stain controls are essential to establish compensation settings, particularly when using biotin-streptavidin systems alongside other fluorophores .

How can I minimize background when using biotin-conjugated PXN antibodies in tissues with endogenous biotin?

Endogenous biotin can significantly interfere with biotin-streptavidin detection systems, particularly in tissues like liver, kidney, and brain. To minimize this interference:

• Block endogenous biotin:

  • Pre-treat tissue sections with avidin-biotin blocking kit

  • Incubate sequentially with avidin solution (binds endogenous biotin), then biotin solution (saturates avidin's biotin-binding sites)

• Alternative detection strategy:

  • Use directly labeled primary antibodies instead of biotin-conjugated antibodies

  • Consider tyramide signal amplification systems if sensitivity is needed

• Sample preparation optimization:

  • Use freshly prepared paraformaldehyde fixative (avoid old solutions that can auto-fluoresce)

  • Optimize antigen retrieval in EDTA buffer (pH 8.0)

  • Include 10% normal serum from the same species as secondary antibody in blocking buffer

Can biotin-conjugated anti-PXN antibodies be effectively used in flow cytometry for studying platelet activation?

Yes, biotin-conjugated anti-PXN antibodies can be effectively used for flow cytometric analysis of platelet activation, though the approach requires careful consideration:

• Protocol considerations:

  • Fixation and permeabilization are essential as PXN is an intracellular protein

  • For multiparameter analysis, stain for surface markers (e.g., P-selectin) before fixation/permeabilization

  • Use streptavidin conjugated to a fluorophore with minimal spectral overlap with other markers

• Data interpretation:

  • PXN serves as a negative regulator of platelet activation, so decreased phosphorylation may correlate with increased activation

  • Correlate PXN phosphorylation status with established platelet activation markers

  • Quantify as mean fluorescence intensity (MFI) of biotin-streptavidin signal

• Validation approach:

  • Validate findings with complementary techniques (Western blot, microscopy)

  • Include appropriate positive controls (e.g., agonist-stimulated platelets)

The research by Sakata et al. demonstrated that PXN functions as an intrinsic negative regulator of platelet activation in mice, highlighting its importance in understanding platelet biology and potential therapeutic targets .

How do I resolve contradictory results between phospho-PXN detection using biotin-conjugated antibodies versus direct Western blotting?

When facing discrepancies between detection methods:

• Systematically evaluate technical factors:

  • Antibody specificity: Test with phosphopeptide competition assays for both detection methods

  • Sample preparation: Ensure phosphatase inhibitors are included during lysis

  • Detection sensitivity: Biotin-streptavidin systems typically provide signal amplification compared to direct detection

• Biological considerations:

  • Temporal dynamics: Phosphorylation events are often transient; ensure consistent timing across methods

  • Spatial constraints: Immunostaining detects localized phosphorylation while Western blot averages across the cell population

• Validation approaches:

  • Employ multiple phospho-specific antibodies targeting different phosphorylation sites

  • Use kinase inhibitors or phosphatase treatments as controls

  • Consider mass spectrometry for unbiased phosphorylation site analysis

How can biotin-conjugated PXN antibodies be incorporated into multiplexed imaging approaches?

Biotin-conjugated PXN antibodies can be integrated into advanced multiplexed imaging workflows through several strategies:

• Sequential staining approaches:

  • Apply, image, and strip/quench biotin-conjugated PXN antibodies in cycle 1

  • Conduct subsequent cycles with other markers

  • Align and overlay images computationally

• Spectrally-distinct reporter systems:

  • Combine streptavidin conjugated to spectrally-distinct fluorophores with directly-labeled antibodies

  • Use biotin-conjugated PXN antibodies with streptavidin-quantum dots for stable, narrow emission spectra

• Spatial analysis considerations:

  • Quantify colocalization between PXN and other focal adhesion proteins

  • Measure distances between PXN and interacting partners using super-resolution techniques

  • Track phosphorylation state changes in relation to cellular structures

What are the considerations for using biotin-conjugated phospho-PXN antibodies in cancer research?

When investigating PXN in cancer research contexts using biotin-conjugated antibodies:

• Clinical significance:

  • PXN phosphorylation status correlates with invasiveness in multiple cancer types

  • Different phosphorylation sites (e.g., Y118) may have distinct roles in migration versus proliferation

• Methodological considerations:

  • Patient-derived samples may require optimized fixation protocols

  • Consider tissue microarray approaches for high-throughput analysis

  • Include appropriate cancer and normal tissue controls

• Data interpretation framework:

  • Correlate PXN phosphorylation with clinical parameters and patient outcomes

  • Integrate with other markers of focal adhesion dynamics

  • Consider subcellular localization of phospho-PXN as it may translocate to different compartments in cancer cells

The immunohistochemical analysis shows that PXN can be effectively detected in various human cancer tissues including laryngeal carcinoma, hashimoto thyroiditis, and thyroid papillary carcinoma, making biotin-conjugated antibodies valuable tools for cancer biomarker research .

How might biotin-conjugated PXN antibodies be integrated with emerging single-cell technologies?

The integration of biotin-conjugated PXN antibodies with single-cell technologies represents an exciting frontier:

• Single-cell proteomics applications:

  • Mass cytometry (CyTOF) using biotin-conjugated antibodies with metal-labeled streptavidin

  • Spatial proteomics platforms to correlate PXN phosphorylation with cellular positioning in tissue architecture

  • Microfluidic antibody-based single-cell Western blotting

• Multi-omics integration:

  • Coupling phospho-PXN detection with single-cell transcriptomics

  • Correlating phosphorylation status with gene expression at individual cell level

  • Developing computational approaches to integrate protein modification data with transcriptional profiles

• Technical adaptations required:

  • Optimization of fixation and permeabilization for single-cell suspension compatibility

  • Careful titration to minimize background in reduced-volume reactions

  • Development of multiplexed detection systems compatible with biotin-streptavidin amplification

By leveraging these emerging technologies, researchers can gain unprecedented insights into the heterogeneity of PXN expression and phosphorylation across cell populations, potentially revealing subpopulations with distinct signaling profiles relevant to disease progression or treatment response.