Bioluminescence Resonance Energy Transfer (BRET)-Based Assay for Measuring Interactions of CRAF with 14-3-3 Proteins in Live Cells

The primary focus of our research is to uncover new vulnerabilities in the RAS-MAP kinase pathway through the study of rare disease associated mutations within. We recently developed another BRET based assay for measuring the auto inhibitory activities of RAF kinases in live cells. Importantly, this assay was essential for characterizing a group of rare germline mutations in the B-RAF cysteine rich domain, which demonstrated the critical importance of this domain for maintaining B-RAF auto inhibition and for preventing constitutive B-RAF biological activity.

This assay measures the interactions of 1433 proteins with the RAF kinase C-RAF, and these interactions play essential roles in mediating RAF function in both development and disease. In addition, this assay combined with previously established assays for measuring RAF auto inhibition and the RAS RAF interaction form the basis of a toolkit for dissecting the regulatory mechanisms of RAF kinases in live cells. To begin, take 293 FT cells cultured in a 10 centimeter dish, and aspirate the media.

Wash cells with five milliliters of PBS. Then add one milliliter of trypsin EDTA and incubate for three to five minutes at 37 degrees Celsius to detach cells from the dish. After incubation, add nine milliliters of complete DMEM to the cells to neutralize the trypsin.

Pipette up and down repeatedly to generate a homogenous single cell suspension, and transfer the cells to a 15 milliliter tube. Immediately transfer 20 microliters of cells to a 1.7 milliliter tube, and mix with 20 microliters of trypan blue stain. Count cells using either an automated cell counter or a hemocytometer.

Transfer the appropriate volume of cells to a 50 milliliter tube and dilute in complete DMEM. Add two milliliters of cell suspension to each well of a six well plate. Incubate cells at 37 degrees Celsius and 10%carbon dioxide overnight.

Prior to transfection, dilute nano luciferase C-RAF and 1433 zeta halo plasmids, along with a PC DNA three empty vector in TE buffer. Label a set of sterile 1.7 milliliter tubes. Add 100 microliters of transfection media to each tube along with the expression constructs.

Now add two microliters of transfection reagent and vortex gently to mix. Pulse spin the tubes briefly in a micro centrifuge, and then incubate the tubes at around 25 degrees Celsius for 15 minutes to allow transfection complexes to form. Afterwards, add transfection complexes to cells, dropwise, and incubate at 37 degrees Celsius for 16 to 20 hours to express the halo and nano tagged proteins.

To begin, label three sets of sterile 1.7 milliliter tubes and one set of sterile 15 milliliter conical bottom tubes. Repair a swing bucket centrifuge with 15 milliliter tube inserts and pre-cool to four degrees Celsius. Take the six well cell culture plate with transfected 293 FT cells.

Aspirate the media from the wells. Add 250 microliter of trypsin EDTA and incubate at 37 degrees until cells begin to detach. Then add one milliliter of 10%FBS supplemented assay media to each well and pipette vigorously to create a single cell suspension.

Transfer one milliliter of cell suspension to pre-labeled 15 milliliter tubes. Add one milliliter of 10%FBS supplemented assay media to each of these tubes and invert five times to mix. Then immediately transfer 20 microliters of cell suspension to tube set one.

Now centrifuge the 15 milliliter tubes for five minutes at 250 G in a pre-cool centrifuge. Makes 20 microliters of trypan blue cell stain with cells in set one, and count using a cell counter or hemocytometer. After removing the 15 milliliter tubes from the centrifuge, aspirate media from cell pellets and re-suspend pellets in serum free assay media to create a single cell suspension.

To re-plate cells in 384 well and six well plates, invert the 15 milliliter tubes several times for a homogenous cell suspension, and transfer 500 microliters to 1.7 milliliter tubes in set two and set three. Add 0.5 microliters of DMSO to set two, and 0.5 microliters of Halo 618 ligand to tube set three, and mix well. Transfer the DMSO and ligand positive cell suspensions to separate wells of reagent reservoirs.

Use a multi-channel pipette to transfer 40 microliters of each suspension to the 384 well culture plate. Transfer the remaining cells to fresh six well culture plates for subsequent western blot analysis, and incubate both 384 well and six well plates overnight at 37 degrees Celsius and 5%carbon dioxide. Take serum free assay media pre-warmed at 37 degrees Celsius.

Dilute the thawed nano luciferase substrate one to 100 in serum free assay media, and transfer it to a reagent reservoir. Using a multi-channel pipette, transfer 10 microliters of substrate mixture to each well of the 384 well culture plate. Gently rotate the plate for one minute.

Insert the 384 well plate into a multi-mode plate reader and record the 460 and 618 nanometers emissions from all wells with cells. Calculate the raw BRET ratios for vehicle positive and ligand positive in milli-BRET units, using the given equation. Calculate the corrected BRET ratio by subtracting the vehicle positive BRET ratio from that of the ligand positive.

The nano C-RAF S 259A mutant reduces the BRET signal by approximately 45%and the nano C-RAF S 621 A mutant by around 25%The double mutant nearly eliminates the BRET signal.