RalA Activation Assay Biochem Kit

Product Uses Include

• Analysis of in vivo RalA activation 

Introduction
The Ras family of small GTPases consists of at least 35 related human proteins that include the oncoproteins HRAS, KRAS and NRAS as the founding members (Colicelli 2004).  The Ral proteins, RalA and RalB, share 58% identity to these oncoproteins and 85% identity with each other (Chardin and Tavitian 1986).  Ral proteins play an important role in diverse cellular processes including endocytosis, exocytosis, oncogenesis and the regulation of transcription and cell morphology (Feig 2003)Like other small GTPases, Ral proteins become activated when they switch from the GDP-bound state to the GTP-bound state (Takai, Sasaki and Matozaki 2001), and it is the GTP-bound form that specifically interacts with their downstream effector proteins.   The fact that Ral family effector proteins will specifically recognize the GTP-bound form of the protein  has been exploited experimentally to develop a powerful affinity purification assay that monitors Ral protein activation (Hofer, Berdeaux and Martin 1998).   The assay uses the Ral Binding Domain (RBD) of the effector protein RalBP1.  The RBD protein motif has been shown to bind specifically to the GTP-bound form of Ral.  The fact that the RBD region of RalBP1 has a high affinity for GTP-Ral makes it an ideal tool for affinity purification of GTP-Ral from cell lysates.  The RalBP1-RBD protein supplied in this kit has been expressed as an His-tagged fusion protein in E. coli. and bound  to colored beads. This allows one to “pulldown” GTP-Ral complexed with Ral-BP1-RBD beads.  This assay provides a simple means of analyzing cellular RalA activities in a variety of systems.  The amount of activated RalA is determined by a Western blot using a RalA specific antibody.

Kit contents

The kit contains sufficient materials for 50 assays and includes reagents for positive and negative controls. The following components are included:

1. His-tagged RalBP1-RBD protein on colored agarose beads
2. RalA-specific monoclonal antibody
3. His-tagged RalA protein
4. GTPγS: (non-hydrolyzable GTP analog) (Cat. # BS01)
5. GDP
6. Cell lysis Buffer
7. Wash Buffer
8. Loading Buffer
9. STOP Buffer
10. Protease inhibitor cocktail (Cat. # PIC02)
11. Manual with detailed protocols and extensive troubleshooting guide

Equipment needed

1. SDS-PAGE minigel system and western blotting transfer apparatus