P4H1 Antibody

What is P4HA1 and why is it important in research?

P4HA1 is a component of prolyl 4-hydroxylase, a key enzyme in collagen synthesis composed of two identical alpha subunits and two beta subunits. The encoded protein provides the major catalytic site of the active enzyme. In collagen and related proteins, P4HA1 catalyzes the formation of 4-hydroxyproline that is essential for proper three-dimensional folding of newly synthesized procollagen chains . P4HA1 has gained significant research interest due to its involvement in cancer progression, particularly in triple-negative breast cancer (TNBC) where its expression correlates with short relapse-free survival in patients receiving chemotherapy .

What molecular forms does P4HA1 take in cellular environments?

P4HA1 can exist in multiple forms including heteromers, dimers, or tetramers. Native gel electrophoresis analysis with Coomassie blue staining has shown purified protein presenting as three major bands, representing tetramer, dimer, and single subunit forms . The gene encodes a polypeptide of 517 amino acid residues and a signal peptide of 17 amino acids. The full-length protein has two glycosylation sites and has three isoforms produced by alternative splicing with molecular weights of 61 kDa, 61 kDa, and 59 kDa .

Which applications are P4HA1 antibodies suitable for?

P4HA1 antibodies have been validated for multiple applications including:

It's recommended to titrate the antibody in each testing system to obtain optimal results as performance can be sample-dependent .

What is the optimal protocol for detecting P4HA1 in Western blots?

For Western blot detection of P4HA1:

• Sample preparation: Lyse cells in RIPA buffer containing protease inhibitors

• Load 20-40 μg of total protein per lane

• Separate proteins on 10% SDS-PAGE gel

• Transfer to PVDF membrane (wet transfer recommended)

• Block with 5% non-fat milk in TBST for 1 hour at room temperature

• Incubate with P4HA1 primary antibody at 1:1000-1:2000 dilution overnight at 4°C

• Wash 3 times with TBST, 5 minutes each

• Incubate with species-appropriate HRP-conjugated secondary antibody at 1:5000 dilution for 1 hour at room temperature

• Wash 3 times with TBST, 5 minutes each

• Develop using ECL substrate and image

The expected molecular weight is approximately 61 kDa, consistent with the calculated molecular weight of the protein .

How should P4HA1 immunostaining be optimized for different tissue types?

For optimal immunohistochemical detection of P4HA1:

• Fix tissues in 10% neutral buffered formalin and embed in paraffin

• Section tissues at 4-6 μm thickness

• Deparaffinize and rehydrate sections

• For antigen retrieval, use TE buffer pH 9.0 (recommended) or citrate buffer pH 6.0 as an alternative

• Block endogenous peroxidase with 3% H₂O₂

• Block non-specific binding with 5-10% normal serum

• Incubate with P4HA1 antibody at 1:50-1:500 dilution depending on the specific antibody and tissue type

• Continue with appropriate detection system

P4HA1 expression has been successfully detected in various tissues including breast cancer, pancreatic cancer, testis, endometrium, placenta, kidney, and skin tissues .

What are common issues with P4HA1 antibody detection and how can they be resolved?

Common issues and solutions include:

• High background signal:

  • Increase blocking time/concentration

  • Reduce primary antibody concentration

  • Use more stringent washing conditions

  • Ensure fresh blocking reagents

• Weak or no signal:

  • Optimize antigen retrieval (try both TE buffer pH 9.0 and citrate buffer pH 6.0)

  • Increase antibody concentration

  • Extend primary antibody incubation time

  • Verify protein expression in your sample type

  • Ensure proper storage of antibody (typically -20°C with 0.02% sodium azide and 50% glycerol pH 7.3)

• Multiple bands:

  • Use positive control lysates (e.g., A431 cells, NIH/3T3 cells, or PC-3 cells)

  • Consider the presence of multiple isoforms (61-63 kDa and 57-59 kDa)

  • Use freshly prepared samples to minimize degradation

How can P4HA1 antibody specificity be validated in experimental systems?

To validate P4HA1 antibody specificity:

• Positive controls: Use cell lines known to express P4HA1 (A431, NIH/3T3, PC-3, HEK-293, or HeLa cells)

• Negative controls:

  • Use unimmunized IgG at the same concentration as the primary antibody

  • Include P4HA1 knockout or knockdown samples generated via CRISPR or siRNA

• Peptide competition assay: Pre-incubate the antibody with excess immunogenic peptide before application to samples

• Multiple antibody validation: Use two different antibodies raised against different epitopes of P4HA1

How does P4HA1 activity impact HIF-1α stabilization and what methods can measure this interaction?

P4HA1 influences HIF-1α stabilization by modulating alpha ketoglutarate (α-KG) and succinate levels, which reduces proline hydroxylation on HIF-1α, enhancing its stability in cancer cells. This P4HA1/HIF-1 axis enhances cancer cell stemness, accompanied by decreased oxidative phosphorylation and reactive oxygen species (ROS) levels .

Methods to study this interaction include:

• HRE-luciferase reporter assay: Introducing the hypoxia response element (HRE)-luciferase reporter plasmid in control and P4HA1-silenced cells shows that knockdown of P4HA1 significantly reduces transcription driven by HRE

• ODD-luciferase reporter assay: This construct contains a firefly luciferase gene fused to the hydroxylation-dependent degradation region of HIF-1α, useful for monitoring HIF-1α stability/degradation

• Hydroxylated HIF-1α detection: Using an antibody against hydroxylated HIF-1α (P402) to demonstrate that silencing P4HA1 increases HIF-1α hydroxylation in cancer cells

• qRT-PCR for HIF-1 target genes: Assessing expression of HIF-1 target genes like lactate dehydrogenase A (LDHA) and pyruvate dehydrogenase kinase 1 (PDK1) in control, P4HA1-silenced, and P4HA1-expressing cells

What high-throughput screening methods exist for identifying P4HA1 inhibitors?

A sophisticated high-throughput screening method has been developed for identifying P4HA1 inhibitors by quantifying succinate, a byproduct of P4H-catalyzed hydroxylation:

• Succinate-Glo™ Hydroxylase assay: This bioluminescence-based assay quantifies succinate production and has proven more sensitive than the hydroxyproline colorimetric assay. The approach involves:

  • Purification of C-P4H1 tetramer from eukaryotic expression systems (particularly HEK-293FT cells)

  • Performing hydroxylation reactions with purified enzyme

  • Quantifying succinate production via bioluminescence

Using this method, researchers screened more than 1400 FDA-approved chemicals and identified over 40 compounds with similar or greater inhibitory ability compared to DHB (a known P4HA1 inhibitor). Key findings included Silodosin and Ticlopidine as novel P4HA1 inhibitors, which inhibited enzyme activity in a dose-dependent manner and suppressed collagen secretion and tumor invasion in 3D tissue culture .

How can P4HA1 antibodies be used to study cancer progression and chemoresistance?

P4HA1 antibodies have proven valuable in studying cancer progression and chemoresistance through multiple approaches:

• Expression correlation studies: Immunohistochemical staining with P4HA1 antibodies has shown increased expression in triple-negative breast cancer (TNBC) and HER2-positive breast cancer. Higher P4HA1 expression correlates with shorter relapse-free survival in TNBC patients who received chemotherapy

• Mechanistic studies: P4HA1 expression reduces proline hydroxylation on HIF-1α by modulating α-KG and succinate levels, enhancing cancer cell stemness. This mechanism has been linked to chemoresistance

• In vivo studies: P4HA1 inhibition sensitizes TNBC to chemotherapeutic agents docetaxel and doxorubicin in xenografts and patient-derived models

• Collagen deposition analysis: P4HA1 antibodies can be used to connect enzyme expression with collagen deposition and cancer metastasis

What role does P4HA1 play in collagen-dependent diseases and how can antibodies help characterize these conditions?

P4HA1 is central to collagen synthesis and has been implicated in various collagen-dependent diseases:

• Fibrosis assessment: P4HA1 antibodies can help quantify enzyme expression in fibrotic tissues, providing insights into disease progression

• Cancer invasion and metastasis: P4HA1-mediated collagen deposition contributes to the extracellular matrix remodeling essential for cancer invasion. Antibodies against P4HA1 can help characterize this process in tissue sections

• Therapeutic target evaluation: As novel P4HA1 inhibitors are developed (like Silodosin and Ticlopidine), antibodies are essential for confirming target engagement and monitoring changes in collagen secretion

• Biomarker potential: P4HA1 expression levels detected by antibodies may serve as prognostic biomarkers in certain cancers, particularly TNBC where expression correlates with chemotherapy response