HPGD Antibody, Biotin conjugated

What is HPGD and what cellular functions does it regulate?

HPGD (15-hydroxyprostaglandin dehydrogenase) is a 266 amino acid protein with a molecular mass of approximately 29 kDa that localizes primarily to the cytoplasm. As a member of the Short-chain dehydrogenases/reductases (SDR) protein family, HPGD catalyzes the NAD-dependent dehydrogenation (oxidation) of hydroxylated polyunsaturated fatty acids, particularly eicosanoids and docosanoids including prostaglandins, lipoxins, and resolvins. This enzymatic activity yields corresponding keto (oxo) metabolites, effectively inactivating these bioactive lipid mediators .

HPGD functions as a critical metabolic enzyme for prostaglandins and serves as a physiological antagonist to COX-2, making it central to inflammatory response regulation. Its expression is particularly notable in colon epithelium, and research has established its role as a tumor suppressor in colon cancer . HPGD can be used as a marker to identify specific cell populations including Common Myeloid Progenitors (CMP) and Thalamus Splatter Neurons .

What are the standard applications for HPGD antibodies in experimental protocols?

HPGD antibodies are utilized across multiple experimental techniques with varying optimization requirements. Common applications include:

• Western Blot (WB): The most widely documented application, typically showing a specific band at approximately 29 kDa in human samples

• Immunohistochemistry (IHC): Both paraffin-embedded (IHC-p) and frozen sections (IHC-fr)

• Immunocytochemistry (ICC) and Immunofluorescence (IF)

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Flow Cytometry

• Immunoprecipitation (IP)

When selecting an antibody, researchers should verify validation data for their specific application and ensure species cross-reactivity matches their experimental model. Commercial antibodies have demonstrated reactivity with human, mouse, and rat HPGD .

How does biotin conjugation enhance HPGD antibody functionality in detection systems?

Biotin conjugation provides several methodological advantages for HPGD detection, particularly in complex experimental setups:

• Signal amplification: The high-affinity interaction between biotin and streptavidin/avidin (Kd ≈ 10^-15 M) enables significant signal amplification without affecting antibody specificity

• Multi-layer detection systems: Enables construction of detection cascades using streptavidin-conjugated enzymes or fluorophores

• Multiplex compatibility: Facilitates detection of multiple targets simultaneously in co-localization studies

• Reduced background: When properly optimized, can reduce non-specific binding compared to directly conjugated detection systems

For detecting low expression levels of HPGD, such as in early-stage tumorigenesis studies, biotin conjugation can provide the sensitivity necessary to quantify subtle changes in expression levels that might be missed with conventional detection systems.

How do I validate HPGD antibody specificity to ensure experimental reproducibility?

Rigorous validation of HPGD antibody specificity should include these methodological approaches:

• Positive and negative control tissues/cells: Colon epithelium serves as an excellent positive control given its high HPGD expression

• Western blot validation: Confirm detection of the expected 29 kDa band in human liver tissue extracts

• Peptide competition assay: Pre-incubation with synthetic HPGD peptide should abolish specific staining

• siRNA/shRNA knockdown: Reduced signal following HPGD gene silencing confirms specificity

• Overexpression controls: Increased signal in cells transfected with HPGD expression vectors, as demonstrated in research using pcDNA6-Wt-PGDH constructs

• Cross-reactivity assessment: Test against related family members in the SDR protein family

For biotin-conjugated antibodies specifically, include additional streptavidin-only controls to assess potential endogenous biotin interference.

How can HPGD antibodies be utilized to investigate tumor suppressor mechanisms in cancer research?

HPGD functions as a tumor suppressor, particularly in colon cancer, through its antagonistic relationship with COX-2 . Methodological approaches for investigating this mechanism include:

• Immunohistochemical profiling: Compare HPGD expression levels across tumor progression stages using calibrated IHC with biotin-conjugated antibodies

• Functional restoration studies: Follow protocols similar to H358 lung cancer cell models, where stable transfection with pcDNA6-Wt-PGDH expression vectors and subsequent xenograft studies demonstrated tumor suppression

• Prostaglandin metabolism quantification: Couple HPGD immunodetection with prostaglandin quantification to establish correlations between enzyme levels and metabolite concentrations

• Co-expression analysis: Implement dual staining protocols to investigate HPGD/COX-2 expression ratios in tumor microenvironments

These approaches can be further enhanced using biotin-conjugated antibodies, particularly for multiplexed detection systems that simultaneously visualize HPGD alongside other pathway components.

What methodological considerations apply when studying HPGD regulation by transcription factors?

Research has established that HPGD is regulated by transcription factors like HNF3β . When investigating these regulatory relationships:

• Chromatin Immunoprecipitation (ChIP): Using biotin-conjugated HPGD antibodies combined with transcription factor antibodies can help identify protein-DNA interactions at the HPGD promoter

• Electrophoretic Mobility Shift Assay (EMSA): Follow protocols similar to those used with H358-HNF3β (tet-off) cells, where nuclear extracts were collected after doxycycline withdrawal and DNA-protein complexes were analyzed using 6% DNA retardation gel

• Expression correlation studies: Implement time-course experiments tracking HPGD upregulation following transcription factor activation, using real-time RT-PCR to quantify temporal relationships

• Reporter gene assays: Construct HPGD promoter-reporter constructs to directly assess transcription factor effects on promoter activity

Research has demonstrated that HNF3β induction can increase HPGD mRNA levels by 10-15 fold within 72-96 hours in appropriate model systems .

How can HPGD antibodies help elucidate mechanisms of drug resistance in hormone-dependent cancers?

HPGD downregulation has been implicated in tamoxifen resistance in breast cancer . Research methodologies to investigate this relationship include:

• Comparative expression analysis: Implement western blot and real-time PCR protocols to quantify HPGD downregulation in resistant versus sensitive cell lines, as demonstrated in studies of TAMr MCF-7 cells

• Reconstitution experiments: Restore HPGD expression in resistant cells to assess drug sensitivity recovery, following protocols that demonstrated restoration of tamoxifen sensitivity to approximately 60% of parental MCF-7 cell response

• Mechanistic pathway analysis: Couple HPGD detection with estrogen receptor signaling pathway components to identify interaction nodes

• Time-course studies: Track HPGD expression changes during resistance development using biotin-conjugated antibodies for enhanced sensitivity

These approaches have revealed that HPGD overexpression can resensitize tamoxifen-resistant breast cancer cells to both tamoxifen and estrogen .

What technical approaches can resolve contradictory data when studying HPGD expression patterns?

When faced with conflicting HPGD expression data across experimental systems:

• Isoform-specific detection: Design experiments accounting for the five known HPGD isoforms by selecting antibodies with validated epitope specificity

• Post-translational modification analysis: Implement immunoprecipitation with biotin-conjugated HPGD antibodies followed by mass spectrometry to identify modifications affecting function or detection

• Subcellular localization studies: Use fractionation protocols coupled with HPGD immunodetection to resolve potential compartmentalization differences

• Context-dependent regulation assessment: Compare HPGD expression across different microenvironments, as exemplified by studies showing variable HPGD responses to estrogen in different cell line models

Careful experimental design with appropriate controls can help reconcile apparently contradictory findings by identifying context-specific regulatory mechanisms.

What are the optimal western blot conditions for detecting HPGD with biotin-conjugated antibodies?

For optimal western blot detection of HPGD using biotin-conjugated antibodies, follow these methodological guidelines:

• Sample preparation: Process human liver tissue (positive control) under reducing conditions using Immunoblot Buffer Group 8 components

• Gel separation: Use 10-12% SDS-PAGE for optimal resolution around the 29 kDa region where HPGD migrates

• Transfer conditions: PVDF membrane transfer at 100V for 1-2 hours in standard transfer buffer provides optimal protein retention

• Blocking protocol: Block with 5% non-fat milk in TBST (Tris-buffered saline with 0.1% Tween-20) for 1 hour at room temperature

• Primary antibody incubation: Dilute biotin-conjugated anti-HPGD antibody to 1 μg/mL in blocking solution and incubate overnight at 4°C

• Detection system: Use HRP-conjugated streptavidin at 1:5000 dilution for 1 hour at room temperature

• Signal development: Enhanced chemiluminescence with exposure times optimized to detect the specific 29 kDa band without saturation

The expected result should show a specific band for HPGD at approximately 29 kDa, consistent with documented findings in human liver tissue .

How should epigenetic regulation of HPGD be investigated using antibody-based approaches?

HPGD expression is regulated through epigenetic mechanisms, including miRNA interactions. To investigate these regulatory mechanisms:

• Combined chromatin and expression analysis: Implement ChIP-seq with HPGD antibodies alongside expression profiling to correlate chromatin states with expression levels

• miRNA-target validation: Design experiments based on identified miRNA-HPGD interactions, as shown in the table below of miRNAs differentially expressed in tamoxifen-resistant cells :

• Dual luciferase reporter assays: Construct reporter systems containing the HPGD 3′-UTR to validate miRNA binding and functional consequences

• Biotin pull-down assays: Use biotinylated miRNAs to capture HPGD mRNA, confirming direct interactions

• Anti-AGO2 RNA immunoprecipitation: Precipitate miRNA-mRNA-protein complexes to identify endogenous miRNA-HPGD interactions

These approaches can help establish the mechanistic basis for epigenetic HPGD regulation in various biological contexts.

What protocols maximize immunohistochemical detection of HPGD in tissue samples?

For optimal immunohistochemical detection of HPGD in tissue samples:

• Fixation protocol: 10% neutral buffered formalin for 24 hours preserves HPGD antigenicity while maintaining tissue architecture

• Antigen retrieval: Heat-induced epitope retrieval using citrate buffer (pH 6.0) for 20 minutes at 95°C

• Blocking considerations:

  • For biotin-conjugated antibodies: Include an avidin/biotin blocking step to reduce endogenous biotin interference

  • Block endogenous peroxidase with 3% hydrogen peroxide for 10 minutes

  • Block non-specific binding with 5% normal serum from the same species as the secondary antibody

• Primary antibody incubation: Optimize dilution (typically 1:100 to 1:500) through titration experiments; incubate overnight at 4°C

• Detection system for biotin-conjugated antibodies: Implement streptavidin-HRP or streptavidin-fluorophore systems with appropriate amplification

• Counterstaining: Light hematoxylin counterstain for brightfield detection; DAPI for fluorescence applications

• Multi-antigen detection: For co-expression studies, design sequential staining protocols to prevent crossreactivity

These protocols should be validated in tissues with known HPGD expression, such as colon epithelium, before application to experimental samples .

How can HPGD antibodies be implemented in xenograft tumor models?

For effective implementation of HPGD antibodies in xenograft studies:

• Pre-implantation cell characterization: Confirm HPGD levels in cells before implantation using western blot and enzymatic activity assays

• Experimental design:

  • Use stable transfection approaches with HPGD expression vectors (pcDNA6-Wt-PGDH) or control vectors (pcDNA6-EV)

  • Select stable cell pools using appropriate selection markers (e.g., 10 μg/ml blasticidin)

  • Implant 5 × 10^6 cells resuspended in 200 μl serum-free medium subcutaneously in athymic mice

• Tumor monitoring protocol: Measure tumor dimensions weekly, calculating volume using the formula: Volume = (width)^2 × length/2

• Endpoint analysis:

  • Process harvested tumors for both IHC (HPGD expression patterns) and biochemical analysis

  • Use biotin-conjugated HPGD antibodies for enhanced signal detection in fixed tumor sections

  • Correlate HPGD expression with tumor growth characteristics and prostaglandin levels

This approach has been validated in studies showing that HPGD expression can suppress tumor growth in appropriate model systems .

What are the methodological considerations for studying HPGD in drug resistance development?

When investigating HPGD's role in drug resistance:

• Resistance model development: Establish resistant cell lines through continuous exposure to therapeutic agents, as exemplified by TAMr MCF-7 cell lines cultured in 100 nM tamoxifen for 12-21 months

• Temporal expression analysis: Track HPGD expression changes throughout resistance development using quantitative western blot and real-time PCR

• Functional reconstitution studies: Implement stable transfection of HPGD in resistant cells to assess functional consequences on drug sensitivity

• Response quantification: Design dose-response experiments to quantify sensitivity restoration following HPGD reintroduction, with expectations of partial restoration (approximately 60% of parental response)

• Mechanistic pathway analysis: Couple HPGD studies with analysis of estrogen responsiveness and alternative resistance pathways

These approaches have demonstrated that HPGD downregulation contributes to tamoxifen resistance, and its restoration can partially reverse this phenotype .