Inhibition of Phospho Raf-1 (Ser259)

HeLa cells were seeded in a 96-well plate(40,000 cells/well) in complete medium, and incubated overnight at 37°C, 5% CO2. The cells were treated for 24 hours with increasing concentrations of Geldanamycin prepared in serum free medium containing 1% FBS.

After treatment, the cells were lysed with 100µL of lysis buffer for 10 minutes at RT with shaking (350 rpm). Raf-1 Phospho (Ser259) and Total levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate(approximately 4,000 cells) were transferred into a 384-well white OptiPlate, followed by 5µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision Nexus™ using standard AlphaLISA settings.

As expected, Geldanamycin (HSP90 inhibitor, disrupts the Raf-1-HSP90 complex) triggered a dose-dependent decrease in the levels of Raf-1 Phospho (Ser259) and Total.

Selectivity of Phospho Raf-1 (Ser259) assay

Raf-1 or Raf-1 p-S259 peptides were titrated into a fixed dilution of positive control lysate (HeLa cells treated with 200 ng/mL PMA). RAF1 Phospho (Ser259) levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of peptide mix was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision Nexus using standard AlphaLISA settings.

Phospho Raf-1 (Ser259) levels were assessed in HeLa cells (WT) and Raf-1 knockout (KO) HeLa cells. Raf-1 KO cells (Abcam ab264978) and WT-HeLa cells were cultured to confluency in T25 flasks in complete medium at 37°C, 5% CO2.

Each flask was lysed in 2 mL of lysis buffer for 10 minutes at RT with shaking (350 rpm). Phospho Raf-1 (Ser259) levels were then evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision Nexus using standard AlphaLISA settings. As expected, Phospho Raf-1 (Ser259) was detected in the WT-Hela cells but not in the Raf-1 KO cell line.