RayBio® Human and Mouse Phospho-mTOR (Ser2448) ELISA Kit. This assay semi-quantitatively measures mTOR phosphorylated at Serine-2448 in cell lysate samples.
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-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
Protocol Outline
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.
Typical Data
Positive Control
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.Â
Storage/Stability
Upon receipt, the kit should be stored at –20°C. Please use within 6 months from the date of shipment.
Aggarwal, H., et al. "Aegle marmelos differentially affects hepatic markers of glycolysis, insulin signalling pathway, hypoxia, and inflammation in HepG2 cells grown in fructose versus glucose-rich environment." Molecular and cellular biochemistry 438.1-2 (2018): 1-16.
Excellent product. Easy to use. We used this kit to measure mTOR phosphorylation in mice hippocampus. It worked much better than the previous kit we tried.
