P3H2 Antibody, Biotin conjugated

What is P3H2 and what cellular functions does it serve?

P3H2 (Prolyl 3-hydroxylase 2, also known as LEPREL1) is a member of the leprecan family of proteins that shows prolyl 3-hydroxylase activity. It catalyzes the post-translational formation of 3-hydroxyproline in -Xaa-Pro-Gly- sequences in collagens, with specificity for types II, IV, and V collagen . P3H2 plays a critical role in the hydroxylation of specific proline residues within the collagen triple helix structure, which is essential for proper collagen folding, stability, and function . As part of the post-translational modification machinery, P3H2 contributes to the extracellular matrix integrity by ensuring correct collagen structure.

What are the molecular characteristics of P3H2 protein?

P3H2 protein has the following molecular characteristics:

• Calculated molecular weight: 81 kDa (708 amino acids) or 60 kDa (527 amino acids)

• Observed molecular weight: 80 kDa in Western blot applications

• Gene ID (NCBI): 55214

• UniProt ID: Q8IVL5

• Full name: Leprecan-like 1

• Alternative names: Prolyl 3-hydroxylase 2, Myxoid liposarcoma-associated protein 4

How does P3H2 Antibody, Biotin conjugated differ from unconjugated P3H2 antibodies?

P3H2 Antibody, Biotin conjugated contains a biotin molecule covalently attached to the antibody, providing enhanced detection capabilities compared to unconjugated antibodies. The biotin conjugation enables:

• Increased sensitivity in detection systems using avidin/streptavidin-based detection methods

• Amplification of signal strength in low-abundance target applications

• Compatibility with multiple secondary detection systems

• Greater flexibility in experimental design compared to unconjugated antibodies

When selecting between conjugated and unconjugated antibodies, researchers should consider their specific detection system requirements and the abundance of their target protein.

What are the validated applications for P3H2 Antibody, Biotin conjugated?

Based on validation data, P3H2 Antibody, Biotin conjugated is primarily optimized for:

• ELISA (enzyme-linked immunosorbent assay)

• Western Blot (WB): Recommended dilution 1:1000-1:4000

• Immunoprecipitation (IP): 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

• Immunohistochemistry (IHC): Recommended dilution 1:50-1:500

• Flow Cytometry

• Immunofluorescence (IF)

• Immunocytochemistry (ICC)

For optimal results with biotin-conjugated antibodies, researchers should validate the antibody in their specific experimental system.

What is the recommended experimental workflow for P3H2 protein detection using this antibody?

The optimal workflow for P3H2 protein detection using biotin-conjugated antibody involves:

• Sample preparation:

  • For cell lysates: Use radioimmune precipitation assay (RIPA) buffer containing protease inhibitors

  • Employ freeze-thaw cycles (typically three) with occasional vortexing

  • Clear lysates of insoluble material by centrifugation at 10,000 × g

• Application-specific protocols:

  • For ELISA: Follow standard indirect ELISA protocol with streptavidin-HRP for detection

  • For other applications: Validate and optimize dilutions based on sample type and detection system

• Detection systems:

  • Streptavidin-conjugated enzymes (HRP/AP) for colorimetric detection

  • Streptavidin-conjugated fluorophores for fluorescence-based detection

When developing experimental workflows, researchers should carefully optimize antibody concentration, incubation times, and washing steps for their specific application.

What tissue and cell types have been validated for P3H2 expression analysis?

P3H2 expression has been successfully detected in the following tissues and cell types:

Human tissues:

• Liver tissue

• Kidney tissue

• Placenta tissue

• Testis tissue

• Skin tissue

• Brain tissue

• Lung tissue

Cell lines:

• L02 cells

• HEK-293 cells

• RCS-LTC cell line

• SAOS-2 cell line

• Human chondrosarcoma cell line (CH1.2)

• Human breast cancer cell lines (MDA 231, MDA 361)

Mouse tissues:

• Placenta tissue

• Kidney tissue

Researchers should note that P3H2 expression levels vary significantly between tissues, with higher expression typically observed in collagen-rich tissues.

What are the common technical challenges when using P3H2 Antibody, Biotin conjugated and how can they be addressed?

For reliable results, researchers should optimize blocking conditions, antibody dilutions, and incubation times for their specific experimental setup.

How should P3H2 Antibody, Biotin conjugated be stored and handled for maximum stability and performance?

For optimal performance and longevity:

• Store at -20°C

• Stable for one year after shipment when stored properly

• Aliquoting is unnecessary for -20°C storage

• Contains 0.03% Proclin 300 as preservative

• Formulated in 50% glycerol, 0.01M PBS, pH 7.4

When working with the antibody:

• Avoid repeated freeze-thaw cycles

• Thaw aliquots completely before use

• Mix gently before pipetting

• Return to -20°C immediately after use

Following these storage and handling guidelines will help maintain antibody performance over time.

How can P3H2 Antibody, Biotin conjugated be utilized in studying P3H2's role in collagen post-translational modifications?

Advanced research applications for studying P3H2's role in collagen modifications include:

• Mapping 3-hydroxyproline sites: The biotin-conjugated antibody can be used in conjunction with mass spectrometry to identify specific sites of 3-hydroxyproline modification in various collagen types.

• Collagen interaction studies: Researchers can employ this antibody to investigate how 3-hydroxylation mediated by P3H2 affects interactions between collagen IV and binding partners such as glycoprotein VI and nidogens 1 and 2 .

• Co-immunoprecipitation: When combined with other techniques, this antibody can help elucidate the interaction network of P3H2 with other proteins involved in collagen modification.

• Quantitative analysis: By using biotin-conjugated antibody in quantitative assays, researchers can measure changes in P3H2 levels under different physiological or pathological conditions.

These approaches provide insights into the fundamental biology of collagen modification and the specific role of P3H2 in this process.

What is the current understanding of P3H2's role in cancer biology, and how can this antibody contribute to this research?

P3H2 has emerged as a potential tumor suppressor, particularly in breast cancer:

• Epigenetic silencing: P3H2 expression is down-regulated in breast cancer through aberrant CpG methylation in the 5' regulatory sequences. Methylation of P3H2 appears to be breast cancer-specific, as no methylation was detected in other epithelial cancers, primary brain tumors, or malignant melanoma .

• Association with cancer subtypes: P3H2 methylation is strongly associated with estrogen-receptor-positive breast cancers, suggesting it may be a breast-cancer-specific tumor suppressor .

• Functional studies: Ectopic expression of P3H2 in cell lines with silenced endogenous gene results in suppression of colony growth, supporting its tumor suppressor role .

This biotin-conjugated antibody can contribute to cancer research by:

• Enabling quantitative assessment of P3H2 protein levels in tumor samples

• Facilitating the screening of breast cancer subtypes for P3H2 expression

• Supporting functional studies examining the mechanism of P3H2's tumor suppressor activity

• Helping identify potential biomarkers based on P3H2 expression patterns

How can P3H2 knockdown/knockout models be validated using this antibody?

For rigorous validation of P3H2 knockdown/knockout models:

• Western blot validation: The antibody can be used to confirm protein reduction/absence in knockdown/knockout models. For example, a marked knockdown in P3H2 protein was observed when cell lysates were probed with P3H2-specific antibody, clearly demonstrating the efficiency of siRNA-mediated knockdown .

• Complementary approaches:

  • Combine protein detection with qRT-PCR to verify decreased mRNA levels

  • Use immunohistochemistry to confirm tissue-specific knockdown patterns

  • Employ functional assays to correlate phenotypic changes with P3H2 reduction

• Controls and standards:

  • Include appropriate loading controls (e.g., proliferating cell nuclear antigen)

  • Use wild-type samples as positive controls

  • Include multiple clones or treatments to account for variability

This methodological approach ensures that research findings can be confidently attributed to the specific loss of P3H2 function.

How does P3H2 Antibody, Biotin conjugated compare with other P3H family antibodies for research applications?

When selecting between P3H family antibodies, researchers should consider:

• The specific P3H family member of interest

• Required applications and detection sensitivity

• Target tissue or cell type

• Experimental design constraints

What methodological considerations should researchers account for when comparing data generated with different P3H2 antibody formats?

When comparing data from different antibody formats:

• Epitope differences: Different antibodies may recognize distinct epitopes on P3H2, potentially affecting detection of specific isoforms or post-translationally modified forms.

• Sensitivity variations: Biotin-conjugated antibodies typically offer higher sensitivity due to signal amplification; researchers should normalize data accordingly when comparing with unconjugated antibodies.

• Background considerations: Different formats may produce varying background signals that require format-specific optimization.

• Cross-platform standardization:

  • Include common positive and negative controls across experiments

  • Use recombinant standards for quantitative comparisons

  • Apply appropriate normalization techniques

• Application-specific issues:

  • For WB: Compare band patterns and molecular weights

  • For IHC/ICC: Evaluate staining patterns and subcellular localization

  • For ELISA: Establish standard curves for each antibody format

Proper methodological documentation and standardization enable valid comparisons across antibody formats and experimental conditions.

What emerging research questions about P3H2 could be addressed using this antibody?

Several promising research directions can be explored:

• Collagen modification landscape: How does P3H2-mediated 3-hydroxylation interact with other post-translational modifications to regulate collagen structure and function?

• Tissue-specific roles: What are the tissue-specific functions of P3H2, particularly in tissues with high expression levels like liver, kidney, and placenta?

• Cancer biology: Beyond breast cancer, what is P3H2's potential role in other cancer types, and how does its loss contribute to cancer progression?

• Biomarker development: Can P3H2 expression levels or activation patterns serve as biomarkers for disease progression or treatment response?

• Therapeutic targeting: Can modulation of P3H2 activity be leveraged for therapeutic interventions in cancer or collagen-related disorders?

These research questions highlight the multifaceted role of P3H2 in normal physiology and disease states.

How can researchers integrate P3H2 antibody-based detection with other -omics approaches for comprehensive pathway analysis?

Integration of antibody-based detection with -omics approaches:

• Multi-omics integration strategies:

  • Combine proteomics data on P3H2 interactome with transcriptomics to identify co-regulated genes

  • Correlate P3H2 protein levels with epigenomic data on methylation status

  • Integrate metabolomics to explore how P3H2 activity affects collagen metabolism

• Spatial -omics approaches:

  • Use biotin-conjugated antibodies for spatial proteomics to map P3H2 distribution

  • Correlate with spatial transcriptomics data to identify location-specific expression patterns

• Systems biology frameworks:

  • Develop network models incorporating P3H2 protein interactions

  • Apply pathway enrichment analysis to identify biological processes affected by P3H2

  • Use machine learning approaches to identify patterns in P3H2-associated data

• Temporal profiling:

  • Track changes in P3H2 levels across development or disease progression

  • Correlate with dynamic changes in the collagen modification landscape

These integrated approaches provide a more comprehensive understanding of P3H2's biological roles and regulatory networks.