Protein Name & Synonyms:
Serine/threonine-protein kinase mTOR (EC 18.104.22.168) (FK506-binding protein 12-rapamycin complex-associated protein 1) (FKBP12-rapamycin complex-associated protein) (Mammalian target of rapamycin) (mTOR) (Mechanistic target of rapamycin) (Rapamycin and FKBP12 target 1) (Rapamycin target protein 1)
- AMPK Signaling
- PI3K-AKT Signaling
This ELISA kit recognizes Human
Compatible Sample Types:
- Cell Lysates
- Tissue Lysates
Method of Detection:
1, 2, or 5 x 96-Well Strip Microplate Kit
- Rapidly measure phosphorylated protein in lysates
- Screen numerous different cell lysates without performing a Western Blot analysis
- Minimal hands-on time, convenient, and non-radioactive material
- Pre-Coated 96-well Strip Microplate
- Wash Buffer
- Anti-Phospho Antibody
- HRP-Conjugated Secondary Antibody
- 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
- 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 1X 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.
A431 cells were treated with recombinant human EGF at 37°C for 20 min. Cells were solubilzed at 4 x 107
cells/ml in Cell Lysate Buffer. Serial dilutions of lysates were analyzed in this ELISA (see Reagent Preparation step 4).
FBS Stimulation of 293 Cell Lines
293 cells were untreated or treated with 20% FBS for 20 min. Cell lysates were analyzed using this phosphoELISA. A431 cells were treated with recombinant human EGF at 37°C for 20 min. Cell lysates were analyzed using Western Blot.
Upon receipt, the kit should be stored at –20°C. Please use within 6 months from the date of shipment.