A. Reagent Preparation
- Bring all microwell strips to room temperature before use.
- Prepare 1X Wash Buffer by diluting 20X Wash Buffer (included in each kit) in Milli-Q or equivalently purified water.
- Dilute the 10X Cell Lysis Buffer #9803 to 1X in Milli-Q or equivalently purified water. 1 mM phenylmethylsulfonyl fluoride (PMSF) should be added fresh each time. This buffer can be stored at 4°C for short-term use (1–2 weeks).
B. Cell Lysate Preparation
NOTE: This procedure is for a 96-well tissue culture plate. It can be modified for other tissue culture plates (6, 12, 24, or 48 well).
- Plate cells of interest in 96-well plate (typically between 6–100 X 103 cells/well) and incubate overnight under appropriate cell culture conditions.
- Rinse cells with 200 μl warm PBS, then add test compounds in serum free mediums and incubate cells for the desired time period.
- Rinse cells twice with 200 µl ice cold PBS, and then add 100 μl/well 1X lysis buffer, keep cells on ice for 5 to 10 minutes.
NOTE: If cell debris is observed it can be removed by brief centrifugation of the plate and transfer of the clear lysates to a new 96 well plate.
- Bring all kit components to room temperature.
- Add 50 µl of the HRP-linked target solution and 50 μl sample to the antibody-coated assay plate. Cover the plate and incubate at room temperature for 3 hours on a horizontal orbital plate shaker.
- Discard plate contents and wash wells 4 times with 200 μl /well of 1X Wash Buffer. Make sure to discard all liquid after each wash but do not allow wells to completely dry.
- Add 100 μl TMB substrate.
- Incubate for 30 minutes at room temperature.
NOTE: Watch the color as it being developed since it may be necessary to stop the reaction before 30 minutes.
- Add 100 μl STOP solution.
- Measure absorbance at 450 nm (for optimal results, read the plate within 30 minutes after adding STOP solution).
NOTE: To determine the absolute amount of the tested substance, a standard curve needs to be generated each time. Please follow the detailed protocol on the product datasheet to determine the concentration range for the standard curve.