A. Reagent Preparation

  1. Bring all microwell strips to room temperature before use.
  2. Prepare 1X Wash Buffer by diluting 20X Wash Buffer (included in each kit) in Milli-Q or equivalently purified water.
  3. 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).

  1. Plate cells of interest in 96-well plate (typically between 6–100 X 103 cells/well) and incubate overnight under appropriate cell culture conditions.
  2. Rinse cells with 200 μl warm PBS, then add test compounds in serum free mediums and incubate cells for the desired time period.
  3. 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.

C. Assay

  1. Bring all kit components to room temperature.
  2. 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.
  3. 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.
  4. Add 100 μl TMB substrate.
  5. 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.

  1. Add 100 μl STOP solution.
  2. 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.

posted April 2010

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