Sc 520

An HRAS activation assay was performed using a GST-fusion protein of the RAS-binding domain (RBD) of RAF1, as instructed by the manufacturer (Pierce Biotechnology). Briefly, SSCs maintained in growth media were washed with cold TBS and lysed. Active RAS was pulled down with GST-RAF1-RBD along with glutathione agarose resin, followed by Western blot detection with an anti-Hras antibody (sc-520, Santa Cruz Biotechnology).

We generated protein array analysis to quantify active Ras by using a mouse antibody (Pan-Ras, OP40, Calbiochem) and GST-tagged Ras-binding-domain (RBD) of Raf (GST-Raf-RBD 1–149) [56 (link)] for Ras detection. To generate the pGex plasmid harboring the GST-tagged Raf-RBD we cloned the Raf-RBD into the pGex vector via BamHI and EcoRI sites. The pGex-GST-Raf-RBD was expressed in BL21 bacteria and the fusion protein was purified using Gluthatione Sepharose beads (GE Healthcare). To spot the eluted fraction, it was diluted with PBS and glycerol. The spotting was performed as described in the previous paragraph. Cell lysates were diluted 1:10 with Array Buffer PLUS. For calibration positive and negative control samples with gammaS-GTP and GDP loading were used. Prior to incubation, the slides were blocked with LiCor Blocking Buffer for 2–6 h. Samples and calibrator-solutions were incubated on the slides shaking overnight. All incubations were performed at 4°C. The slides were then washed with array buffer and incubated with specific rabbit-derived detection Ras antibody sc-14022 and sc-520 (Santa Cruz Biotechnology). Slides were subsequently treated as described in the previous paragraph.