Preparation of Test Samples

The cell signaling Flex Sets are designed to measure total or phosphorylated proteins from denatured cell lysate samples. The tested cells need to be lysed and denatured using the 5X denaturation buffer (provided in the kit) before used in a Flex Set assay. The standard curve for each Flex Set covers a defined set of concentrations between 3.9 and 1000 U/mL. It may be necessary to dilute test samples to ensure that their mean fluorescence values fall within the limits or range of the generated standard curve. For best results, samples that are known or assumed to contain high levels of a given protein should be diluted. In cases where the samples are known or assumed to contain low levels of a given protein, the sample should be lysed in a lower volume of lysis buffer thereby concentrating the protein in the sample. It is important that the cell number or the total protein concentration of the cell lysate sample is known so that results determined using the Flex Sets can be normalized (e.g., U/mL/106 cells or U/mL/pg of cell lysate). It is necessary to heat the 5X denaturation buffer to 37°C before use (shake or vortex until all precipitates have gone back into solution). The final concentration of the denaturation buffer should reach 1X once mixed with cells to achieve denaturation of the cell lysate.

3.3.2.1. Cells in Suspension

1. Count cells in sample. This is to give an approximate idea of protein concentration, which should be greater than 1 mg/mL (protein concentration is dependent on cell type, e.g., Jurkat = 100 - 200 pg/106 cells whereas peripheral blood lymphocytes = 25 - 50 pg/106 cells).

2. Treat cells to induce or inhibit protein phosphorylation as required for the experiment.

3. Add appropriate amount of 5X denaturation buffer so that the final concentration is 1X. Alternatively, ice-cold PBS can be added to the tube and the cells pelleted. Add an appropriate amount of 1X denaturation buffer (prepared by diluting the 5X denaturation buffer with water) to resuspend the cell pellet.

4. Immediately place in a boiling water bath for 5 min.

5. Determine protein concentration.

6. Dilute cell lysate sample by the desired dilution factor (i.e., 1:2, 1:10, or 1:20) using the appropriate volume of assay diluent. Sample must be diluted at least 1:2 to reduce the percentage of sodium dodecyl sulfate.

7. Mix sample dilutions thoroughly before transferring samples to the appropriate assay tubes containing capture beads.

3.3.2.2. Adherent Cells

1. Count cells before plating. This is to give an approximate idea of protein concentration, which should be greater than 1 mg/mL.

2. Treat cells to induce or inhibit protein phosphorylation as required for the experiment.

3. Add the appropriate amount of 5X denaturation buffer so that the final concentration is 1X. Alternatively, aspirate off all liquid and add denaturation buffer diluted to 1X with water. Scrape or agitate cells to dislodge from plate.

4. Immediately place in a boiling water bath for 5 min.

5. Determine protein concentration.

6. Dilute cell lysate sample by the desired dilution factor (i.e., 1:2, 1:10, or 1:20) using the appropriate volume of assay diluent. Sample must be diluted at least 1:2.

7. Mix sample dilutions thoroughly before transferring samples to the appropriate assay tubes containing capture beads.

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