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

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

$541.00  
PTE-HIF1A-P402-T-1
+

Antigen Information

Accession Number:
  • Q16665
Target Species:
  • Human

Assay Format

Pathway:
  • Apoptosis
Specificity:
The antibody pair provided in this kit recognizes human hydroxylated HIF-1 alpha hydroxylated at site Proline-402 as well as Hif-1 alpha.
Number of Targets Detected:
2
Species Detected:
  • Human
Compatible Sample Types:
  • Cell Lysates
  • Tissue Lysates
Design Principle:
  • Sandwich-based
Method of Detection:
Colorimetric
Quantitative/Semi-Quantitative:
Semi-Quantitative
Solid Support:
96-well Microplate

Product Specifications

Size:
1, 2, or 5 x 96-Well Strip Microplate Kit

Introduction

RayBio® Hydroxylated-HIF-1alpha (Pro402) and HIF-1alpha 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 and 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 or biotinylated anti-HIF-1alpha antibody is used to detect HIF-1alpha. After washing away unbound antibody, HRP-conjugated anti-rabbit IgG or HRP-conjugated streptavidin 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) or HIF-1alpha bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.

Product Features

  • Simultaneously measure hydroxylated protein and pan protein in one experiment (for normalization purpose)
  • Screen numerous different cell lysates without performing a Western Blot analysis
  • Minimal hands-on time, convenient, and non-radioactive material

Application Notes

Kit Components
  • Pre-Coated 96-well Strip Microplate
  • Wash Buffer
  • Anti-hydroxylated Antibody
  • Anti-Pan Antibody
  • HRP-Conjugated Secondary Antibody
  • Streptavidin-Conjugated HRP
  • Assay Diluent
  • TMB One-Step Substrate
  • Stop Solution
  • Lysis Buffer
  • Positive Control Sample
Other Materials Required
  • Distilled or deionized water
  • 100 ml and 1 liter graduated cylinders
  • Tubes to prepare sample dilutions
  • Protease and Phosphatase inhibitors
  • Precision pipettes to deliver 2 µl to 1 ml volumes
  • Adjustable 1-25 ml pipettes for reagent preparation
  • Benchtop rocker or shaker
  • Microplate reader capable of measuring absorbance at 450 nm
Protocol Outline
  1. Prepare all reagents and samples as instructed in the manual.
  2. Add 100 µl of sample or positive control to each well.
  3. Incubate 2.5 h at RT or O/N at 4 °C.
  4. Add 100 µl of prepared primary antibody to each well.
  5. Incubate 1 h at RT.
  6. Add 100 µl of prepared 1X HRP-Streptavidin to each well.
  7. Incubate 1 h at RT.
  8. Add 100 µl of TMB One-Step Substrate Reagent to each well.
  9. Incubate 30 min at RT.
  10. Add 50 µl of Stop Solution to each well.
  11. Read at 450 nm immediately.

Typical Data

Positive Control
HeLa cells were treated with MG132. Solubilize cells at 4 x 107 cells/ml in Cell Lysate Buffer. Serial dilutions of lysates were analyzed in this ELISA. Please see step 3 of Part VI Reagent Preparation for detail.
STS Stimulation of HeLa Cell Line
HeLa cells were treated or untreated with STS. Cell lysates were analyzed using this ELISA and Western Blot.


Storage/Stability

Upon receipt, the kit should be stored at –20°C. Please use within 6 months from the date of shipment. After initial use, Wash Buffer Concentrate (Item B), Assay Diluent (Item E), TMB One-Step Substrate Reagent (Item H), Stop Solution (Item I) and Cell Lysate Buffer (Item J) should be stored at 4°C to avoid repeated freeze-thaw cycles. Return unused wells to the pouch containing desiccant pack, reseal along entire edge, and store at –20°C. Item D, store at 2-8°C for up to one month (store at -20°C for up to 6 months, avoid repeated freeze-thaw cycles). Reconstituted Positive Control (Item K) should be stored at -70°C.

No posts found

Write A Review

Usage

This product is furnished for LABORATORY RESEARCH USE ONLY.
Not for diagnostic or therapeutic use.

Amazon Gift Card Program

Related Products Related Products