Protein Name & Synonyms:
RAC-alpha serine/threonine-protein kinase (EC 18.104.22.168) (Protein kinase B) (PKB) (Protein kinase B alpha) (PKB alpha) (Proto-oncogene c-Akt) (RAC-PK-alpha)
- Insulin Signaling
- mTOR Signaling
- PI3K-AKT Signaling
The antibodies provided in this kit recognizes human/mouse/rat AKT phosphorylated at Serine-473 and total AKT for comparison.
Number of Targets Detected:
Method of Detection:
1, 2, or 5 x 96-Well Microplate Kit
- Site and signal pathway-specific
- In vitro detection of adherent cell culture
- No sample lysis is needed
- Compatible with a standard ELISA plate reader
- Faster results than with ELISA
- Adaptable for high-throughput screening and drug discovery
- uncoated 96-well Microplate
- Wash Buffer A
- Wash Buffer B
- Fixing Solution
- Quenching Buffer
- Blocking Buffer
- Anti-phospho 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 manufacture'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.
The entire kit may be stored at -20°C for up to 6 months from the date of shipment. Avoid repeated freeze-thaw cycles.