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 lysis buffer. Serial dilutions of lysates were analyzed in this ELISA. Please see step 3 of Part VI. Reagent Preparation for details.
Recombinant Human EGF Stimulation of A431 Cell Lines
A431 cells were treated or untreated with EGF. Cell lysates were analyzed using this phosphoELISA 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.
