Human Hydroxylated-HIF-1alpha (P402) ELISA Kit

ELISA Kits|PTM ELISA|Hydroxylation Assays

Blue ice

RayBio® Human Hydroxylated-HIF-1 alpha (P402) ELISA Kit. This ELISA is for measuring hydroxylated HIF-1 alpha (Pro-402) in human cell lysates.

Sandwich-based

Semi-Quantitative

Pre-Coated 96-well Strip Microplate
Wash Buffer
Anti-Hydroxylated Antibody
HRP-Conjugated Secondary Antibody
Assay Diluent
TMB One-Step Substrate
Stop Solution
Lysis Buffer
Positive Control Sample

Prepare all reagents and samples as instructed in the manual.
Add 100 µl of sample or positive control to each well.
Incubate 2.5 h at RT or O/N at 4°C.
Add 100 µl of prepared primary antibody to each well.
Incubate 1 h at RT.
Add 100 µl of prepared 1
X HRP-Streptavidin to each well.
Incubate 1 h at RT.
Add 100 µl of TMB One-Step Substrate Reagent to each well.
Incubate 30 min at RT.
Add 50 µl of Stop Solution to each well.
Read at 450 nm immediately.

The antibody pair provided in this kit recognizes human Hydroxylated-HIF-1alpha hydroxylated at site Proline-402

RayBio® Hydroxylated-HIF-1alpha (Pro402) ELISA kit is a very rapid, convenient and sensitive assay kit that can monitor the activation or function of important biological pathways in human cell lysates. By determining hydroxylated HIF-1alpha protein in your experimental model system, you can verify pathway activation in your cell lysates. You can simultaneously measure numerous different cell lysates without spending excess time and effort in performing a Western Blotting analysis.This Sandwich ELISA kit is an in vitro enzyme-linked immunosorbent assay for the measurement of human hydroxylated-HIF-1alpha. An anti-pan HIF-1alpha antibody has been coated onto a 96-well plate. Samples are pipetted into the wells and HIF-1alpha present in a sample is bound to the wells by the immobilized antibody. The wells are washed and rabbit anti-Hydroxylated-HIF-1alpha (Pro402) antibody is used to detect hydroxylated HIF-1alpha. After washing away unbound antibody, HRP-conjugated anti-rabbit IgG is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of hydroxylated HIF-1alpha (Pro402) bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.

HeLa cells were treated or untreated with MG132 and DMOG. Cell lysates were analyzed using this ELISA and Western Blot.