PHD1/prolyl hydroxylase Rabbit Recombinant mAb

目录号：A5251

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使用信息

抗体应用

WB,ELISA

稀释比例

1:1000

反应性

Human Mouse Rat

MW (kDa)

44kDa

抗体类型

Rabbit

浓度

1mg/ml

储存液配方

10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide.

储存条件
(自收到货起)

Store at –20°C.

Datasheet & SDS

生物描述

特异性	PHD1/prolyl hydroxylase Rabbit Recombinant mAb detects endogenous level of total PHD1/prolyl hydroxylase.
背景	Prolyl hydroxylases (PHDs) play an important role in regulating HIF-α isoform stability. Three PHD isoforms have been identified in mammals (PHD1, PHD2, and PHD3) and are ubiquitously expressed in most tissues. In normoxic conditions, PHDs act as oxygen-sensing enzymes and promote hydroxylation of HIF-α subunits, allowing recognition by the protein von Hippel-Lindau (pVhl) ubiquitin ligase complex and subsequent proteasomal degradation. PHD activity is reduced during hypoxia, leading to stabilization of HIF-α subunits and their subsequent translocation to the nucleus where they can dimerize with HIF-β, subsequently triggering the transcription of specific target genes.

特异性

PHD1/prolyl hydroxylase Rabbit Recombinant mAb detects endogenous level of total PHD1/prolyl hydroxylase.

背景

Prolyl hydroxylases (PHDs) play an important role in regulating HIF-α isoform stability. Three PHD isoforms have been identified in mammals (PHD1, PHD2, and PHD3) and are ubiquitously expressed in most tissues. In normoxic conditions, PHDs act as oxygen-sensing enzymes and promote hydroxylation of HIF-α subunits, allowing recognition by the protein von Hippel-Lindau (pVhl) ubiquitin ligase complex and subsequent proteasomal degradation. PHD activity is reduced during hypoxia, leading to stabilization of HIF-α subunits and their subsequent translocation to the nucleus where they can dimerize with HIF-β, subsequently triggering the transcription of specific target genes.

实验方法

Western Blotting

Sample preparation

1. Aspirate media from cultures and Wash the cells with 1X PBS.
2. Lyse cells by adding 1X SDS sample buffer and transfer the extract to a microcentrifuge tube. Keep onice.
3. Sonicate for 10–15 sec to complete cell lysis and shear DNA.
4. Heat a 20 µl sample to 95–100°C for 5 min, then cool on ice.
5. Centrifuge for 5 min (with Microcentrifuge).
6. Load appropriate volumes of samples onto SDS-PAGE gel (loading quantity of protein sample depends on the concentration of extracted proteins).
NOTE: At the same time, please load the pre-stained molecular weight markers to determine molecular weights and verify electrotransfer.
7. Electrotransfer to nitrocellulose/PVDF membrane.

Membrane Blocking and Antibody Incubations

a. Blocking

1. (Optional) After transfer, wash the transferred membrane with TBS for 5 min at room temperature.
2. Incubate the membrane in the blocking buffer for 1 hr at room temperature.
3. Wash three times for 5 min each with TBST.

b. Antibodies Incubation

1. Incubate membrane and primary antibody (at the appropriate dilution and diluent recommended) in a primary antibody dilution buffer with gentle agitation overnight at 4°C.
2. Wash three times for 5 min each with TBST.
3. Incubate membrane with an appropriate second antibodydissolved in the blocking buffer with gentle agitation for 1 hr at room temperature.
4. Wash three times for 5 min each with TBST.
5. Proceed with detection.

Detection of Proteins

1. After antibodies incubation, Wash membrane three times for 5 minutes in TBST.
2. PrepareECL Reagent (or other chromogenic agents/substrate according to your second antibody). Mix well.
3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.

参考文献	[1] Thomas A, et al. Sci Rep. 2016, 6:24618.

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