Time-resolved Förster Resonance Energy Transfer Assays for Measurement of Endogenous Phosphorylated STAT Proteins in Human Cells

The TR-FRET based assays provide new cost effective tools for the screening and pharmacological characterization of specific and selective modulators of the JAK/STAT signaling pathways. This technique is much easier, rapid, reproducible, and robust than conventional methods like Western blot and ELISA. No washing steps are required and reagents are added in a single step.

This immunoassay platform can be easily applied to other cell signaling proteins provided specific antibodies are available. To design reproducible assays, you must use good cell culture practices and establish standard operating procedures. In addition, you must carefully optimize the cell culture and treatment conditions.

Demonstrating the procedure will be Genevieve Chatel, a scientist from our laboratory. To begin, culture HeLa and A431 cells in a humidified 37 degree Celsius and 5%carbon dioxide incubator using DMEM supplemented with 10%FBS. When the cells reach 70 to 80%confluence, trypsinize them and either passage or use them for assays.

To perform the assay, dispense 50 microliters of cells at the pre-optimized density into a 96-well tissue culture-treated plate in the appropriate culture medium, then incubate them overnight in a 37 degree Celsius and 5%carbon dioxide incubator. The following day, prepare intermediate twofold and fourfold dilution series of the test compounds by serially diluting the compound in a serum-free medium across 12 wells of a polypropylene 96-well plate. For cell stimulation, add 50 microliters of serum-free medium containing the simulator at a 2X concentration and incubate the cells for the pre-optimized time at either room temperature or 37 degrees.

For cell inhibition, add 25 microliters of serum-free medium containing the inhibitor at a 4X concentration and incubate the cells for the pre-optimized time at either room temperature or 37 degrees, then add 25 microliters of serum-free medium containing the stimulator at a 4X concentration and incubate for the pre-optimized time. Next, to lyse the cells, prepare the 1X supplemented lysis buffer and add phosphatase inhibitor cocktail to it. After carefully removing and discarding the cell culture medium, immediately add 50 microliters of the prepared 1X supplemented lysis buffer and incubate for 30 minutes at room temperature under shaking with moderate agitation.

For TR-FRET detection, prepare the 4X antibody detection mix in 1X detection buffer, then prepare the EUAB1 and FRAB2 antibody solutions, as well as the EUAB3 and FRAB4 antibody solutions in 1X detection buffer. Finally, mix pre-diluted EUAB1 with pre-diluted FRAB2 for detection of phosphoprotein and pre-diluted EUAB3 and pre-diluted FRAB4 for detection of total protein. Then carefully pipette 15 microliters of the cell lysate from the 96-well culture plate to a well of a white low-volume 384-well microplate.

Next, add 15 microliters of the positive control lysate and 15 microliters of 1X lysis buffer as a negative control to separate assay wells. To wells containing 15 microliters of the lysate, add five microliters of the corresponding 4X antibody detection mix to detect the phosphoprotein or total protein. After covering the plate with a plate sealer, incubate it at room temperature for one hour to overnight depending on the assay kit.

After the incubation is complete, remove the adhesive plate sealer and read the plate on a TR-FRET compatible microplate reader. Results of TR-FRET assays for detection and quantification of phospho-STAT1 with total STAT1 and phospho-STAT4 in U266B1 cells along with phospho-STAT5 with total STAT5 in A431 cells are represented using concentration response curves. Similarly, TR-FRET assays for detection and quantification of phospho-STAT3 and total STAT3 and phospho-STAT6 and total STAT6 in HeLa cells are represented using concentration response curves.

Overall, all assays showed robust TR-FRET signals, wide dynamic ranges, low inter-well coefficient variation, and acceptable signal-to-background ratios. Treatment of cells with JAK activators, interferon alfa-2B and IL-4 and EGF showed the anticipated concentration-dependent increase in STAT phosphorylation at specific tyrosine residues, while the corresponding total STAT proteins remained stable. Both JAK inhibitor and erlotinib inhibited the corresponding phospho-STAT levels in a concentration-dependent manner.

Results of phospho-STAT4 stimulation by interferon alfa-2B in a suspension cell line or phospho-STAT6 stimulation by IL-4 in an adherence cell line using the one-plate protocol were consistent with those obtained using the two-plate protocol, thus showing successful adaptability of a two-plate transfer protocol to a one-plate all-in-one well protocol. Results of the intra-plate variability study for the phospho-STAT1 assay using a suspension cell line and the phospho-STAT3 assay using an adherence cell line demonstrate the robustness of these phospho-STAT assays for HTS applications. It is essential to supplement the lysis buffer with the phosphatase inhibitor cocktail to prevent dephosphorylation of the phosphorylated proteins from active phosphatase present in the whole cell extract.