Protein Name & Synonyms:
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)
The antibody pair provided in this kit recognizes human Hydroxylated-HIF-1alpha hydroxylated at site Proline-402
1, 2, or 5 x 96-Well Microplate Kit
- Rapidly measure Hydroxylated protein in lysates
- Simultaneously measure hydroxylated protein and pan protein in one experiment (for normalization purpose)
- No sample lysis is needed
- Compatible with a standard ELISA plate reader
- Uncoated 96-well Strip Microplate
- Wash Buffers
- Fixing Solution
- Quneching Buffer
- Blocking Buffer
- Anti-cleaved Antibody
- Anti-pan antibody
- HRP-Conjugated Secondary Antibody
- TMB One-Step Substrate
- Stop Solution
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
- Seed 10,000-30,000 cells into each well and incubate overnight.
- Apply various treatment, inhibitors or activators according to manufacturer's instructions.
- Add 100 µl of Fixing Solution into each well and incubate for 20 min at RT with shaking.
- Add 200 µl of prepared 1X Quenching Buffer and incubate 20 min at RT.
- Add 200 µl of Blocking Solution and incubate for 1 h at 37°C.
- Add 50 µl of 1X anti-phospho-protein specific antibody or anti-pan-protein specific antibody to each well and incubate for 2 h at RT.
- Add 50 µl of prepared 1X HRP-Anti-Rabbit or Mouse IgG and incubate for 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.
Upon receipt, the kit should be stored at 20°C. Please use within 6 months from the date of shipment.