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Hydroxy-HIF-1α (Pro564) Antibody [P20E16]

Cat.No.: F0384

Application: Reactivity: Human, Monkey

Lane 1: HeLa (MG132, 10 μM, 4 h), Lane 2: HeLa (MG132, 10 μM; DMOG, 1 mM)

Usage Information

Dilution
WB1:1000 IP1:50 CHIP1:3200 - 1:6400

Application
WB, IP, IF

Reactivity
Human, Monkey

Source
Rabbit Monoclonal Antibody

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW
120 kDa

Datasheet & SDS

Biological Description

Specificity
Hydroxy-HIF-1α (Pro564) Antibody [P20E16] detects endogenous levels of HIF-1α only when hydroxylated at Pro564.

Clone
P20E16

Synonym(s)
Hypoxia-inducible factor 1-alpha; HIF-1-alpha; HIF1-alpha; ARNT-interacting protein; Basic-helix-loop-helix-PAS protein MOP1; Class E basic helix-loop-helix protein 78 (bHLHe78); Member of PAS protein 1; PAS domain-containing protein 8; HIF1A; BHLHE78; MOP1; PASD8

Background
Hydroxy-HIF-1α (Pro564) refers to the oxygen-dependent, trans-4-hydroxylated form of proline 564 within the oxygen-dependent degradation domain (ODDD) of the 120 kDa HIF-1α subunit, a basic helix-loop-helix/PAS family transcription factor. This site-specific modification is catalyzed primarily by prolyl hydroxylase domain enzyme 2 (PHD2), which requires oxygen, α-ketoglutarate, Fe²⁺, and ascorbate, and occurs at one of two LXXLAP motifs (Pro402 and Pro564) in the HIF-1α sequence. Hydroxylation at Pro564 introduces conformational rigidity, revealing a hydrophobic core that enables high-affinity binding to the von Hippel-Lindau (VHL) E3 ubiquitin ligase complex, resulting in rapid polyubiquitination and proteasomal degradation of HIF-1α under normoxic conditions. This ensures that HIF-1α levels remain low and hypoxia-responsive genes are not activated when oxygen is adequate. The modification at Pro564, often in concert with Pro402 hydroxylation, acts as a molecular oxygen sensor, tightly controlling HIF-1α stability and blocking its nuclear accumulation and transcriptional activation of hypoxia-response elements (HREs) that regulate angiogenesis, glycolysis, erythropoiesis, and metastasis. Under hypoxia, PHD activity is suppressed, non-hydroxylated HIF-1α escapes degradation, accumulates in the nucleus, and initiates rapid gene induction. Disruption of this regulatory axis, whether through VHL mutations, PHD inhibition, or Pro564 mutation, results in persistent HIF-1α signaling that can drive tumor growth, vascularization, and disease phenotypes, including von Hippel-Lindau syndrome, cancer, pulmonary hypertension, and ischemic responses, with additional regulation by asparaginyl hydroxylation (FIH-1) and oxygen-independent pathways such as PI3K/AKT signaling.

Background

Hydroxy-HIF-1α (Pro564) refers to the oxygen-dependent, trans-4-hydroxylated form of proline 564 within the oxygen-dependent degradation domain (ODDD) of the 120 kDa HIF-1α subunit, a basic helix-loop-helix/PAS family transcription factor. This site-specific modification is catalyzed primarily by prolyl hydroxylase domain enzyme 2 (PHD2), which requires oxygen, α-ketoglutarate, Fe²⁺, and ascorbate, and occurs at one of two LXXLAP motifs (Pro402 and Pro564) in the HIF-1α sequence. Hydroxylation at Pro564 introduces conformational rigidity, revealing a hydrophobic core that enables high-affinity binding to the von Hippel-Lindau (VHL) E3 ubiquitin ligase complex, resulting in rapid polyubiquitination and proteasomal degradation of HIF-1α under normoxic conditions. This ensures that HIF-1α levels remain low and hypoxia-responsive genes are not activated when oxygen is adequate. The modification at Pro564, often in concert with Pro402 hydroxylation, acts as a molecular oxygen sensor, tightly controlling HIF-1α stability and blocking its nuclear accumulation and transcriptional activation of hypoxia-response elements (HREs) that regulate angiogenesis, glycolysis, erythropoiesis, and metastasis. Under hypoxia, PHD activity is suppressed, non-hydroxylated HIF-1α escapes degradation, accumulates in the nucleus, and initiates rapid gene induction. Disruption of this regulatory axis, whether through VHL mutations, PHD inhibition, or Pro564 mutation, results in persistent HIF-1α signaling that can drive tumor growth, vascularization, and disease phenotypes, including von Hippel-Lindau syndrome, cancer, pulmonary hypertension, and ischemic responses, with additional regulation by asparaginyl hydroxylation (FIH-1) and oxygen-independent pathways such as PI3K/AKT signaling.

References
https://pubmed.ncbi.nlm.nih.gov/15104534/ https://pubmed.ncbi.nlm.nih.gov/15304631/

Reference Table for Selecting PVDF Membrane Pore Size Specifications

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%