NRas activation was determined as reported previously [12 (link)]. Briefly, lysates were cleared, and diluted to equal protein concentration as confirmed by Bradford Analysis. Raf-RBD beads (Cytoskeleton) were added to each sample and incubated at 4°C, collected by centrifugation, washed, and resuspended in Laemmli Sample buffer (Bio-Rad). The samples were then run on a Tris-HCl gel, transferred to a nitrocellulose membrane, and immunoblotted with a primary antibody to NRas (Santa Cruz) and appropriate secondary antibody (LiCor). The membrane was visualized using LiCor’s Odyssey infrared imaging system. Ponceau Red staining for total GST-RBD was used as loading control.
Raf rbd beads
Manufactured by Cytoskeleton
Raf-RBD beads are a specialized laboratory product used for the detection and purification of active Ras proteins. They contain the Ras-binding domain (RBD) of the Raf protein, which specifically binds to the active, GTP-bound form of Ras. These beads can be used in various biochemical assays to isolate and study the activity of Ras proteins.
Automatically generated - may contain errors
2 protocols using raf rbd beads
1
Determination of NRas Activation
2
NRAS Activation Analysis Protocol
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