Related Experiment Video
Updated: Jul 5, 2026
09:58
RhoC GTPase Activation Assay
Published on: August 22, 2010
Rac and Rap GTPase activation assays
Ulla G Knaus1, Alison Bamberg, Gary M Bokoch
1Department of Immunology, The Scripps Research Institute, La Jolla, CA, USA.
Methods in Molecular Biology (Clifton, N.J.)
|May 6, 2008
Summary
This study details affinity-based pull-down assays for detecting Ras superfamily GTPase activity, specifically Rac/Cdc42 and Rap, in neutrophils. These methods are crucial for understanding neutrophil signaling pathways.
Area of Science:
- Cellular Biology
- Molecular Biology
- Immunology
Background:
- Ras superfamily GTPase activity is vital for neutrophil signaling.
- Advances in high-affinity probes, often fused to glutathione-S-transferase (GST), facilitate GTPase activation monitoring.
- These probes bind preferentially to the GTP-bound form of small GTPases.
Purpose of the Study:
- To describe affinity-based pull-down assays for detecting specific GTPase activities in neutrophils.
- To provide a method for analyzing signaling events elicited by ligands and cellular processes in neutrophils.
- To enable routine analysis of certain Rho and Ras GTPase family members.
Main Methods:
- Development of probes by fusing high-affinity GTPase-binding effector domains to glutathione-S-transferase (GST).
- Coupling of these GST-effector probes to beads for complex extraction.
- Quantification of active GTP-binding proteins by immunoblotting.
Main Results:
- Successful implementation of affinity-based pull-down assays for Rac/Cdc42 and Rap activity detection.
- Demonstration of routine analysis for specific Rho and Ras GTPase family members.
- Established methodology for monitoring GTPase activation in stimulated neutrophils.
Conclusions:
- Affinity-based pull-down assays are effective for detecting Rac/Cdc42 and Rap activity in neutrophils.
- These assays contribute to a better understanding of neutrophil signaling pathways.
- The described methods advance the study of small GTPase activation in cellular processes.
Related Concept Videos
01:24Small GTPases - Ras and Rho
Ras and Rho are small monomeric GTPases that act downstream of receptor tyrosine kinase (RTK) and regulate various cellular processes. These GTPases switch between active and inactive states by binding to guanine nucleotides.
Three regulatory proteins control their activity:
Three regulatory proteins control their activity:
01:22Activation and Inactivation of G Proteins
Heterotrimeric G proteins are guanine nucleotide-binding proteins. As the name suggests, heterotrimeric G proteins are composed of three subunits: alpha, beta, and gamma. They remain GDP-bound or GTP-bound inside the cells and switch between inactive/active states. The Gα subunit possesses the nucleotide-binding pocket that binds guanine nucleotides and switches between GDP or GTP-bound states. In contrast, the Gꞵ and Gγ subunits are always bound together with high affinity and are together...
02:14GTPases and their Regulation
Guanine nucleotide-binding proteins (G-proteins), also known as GTPases, are a superfamily of proteins that regulate many cellular processes, such as cell signaling, vesicular transport, and the regulation of cell shape and motility. Mutation or dysfunction of these proteins can lead to disease. There are around 40,000 known G-proteins that can broadly be classified into two groups ‒ small G-proteins consisting of a single domain and large multi-domain G-proteins.
Large G-proteins, also known...
Large G-proteins, also known...
02:14GTPases and their Regulation
Guanine nucleotide-binding proteins (G-proteins), also known as GTPases, are a superfamily of proteins that regulate many cellular processes, such as cell signaling, vesicular transport, and the regulation of cell shape and motility. Mutation or dysfunction of these proteins can lead to disease. There are around 40,000 known G-proteins that can broadly be classified into two groups ‒ small G-proteins consisting of a single domain and large multi-domain G-proteins.
Large G-proteins, also known...
Large G-proteins, also known...
01:14Rab Proteins
Rab proteins constitute the largest family of monomeric GTPases, of which 70 members are present in humans. Rab proteins and their effectors regulate consecutive stages of vesicle transport such as vesicle transport, docking, and fusion to the correct recipient membrane.
Rab proteins switch between a cytosolic, GDP-bound inactive state and a membrane-anchored, GTP-bound active state. By themselves, Rabs show slow rates of GDP/GTP exchange and GTP hydrolysis. Thus, Rab proteins are considered...
Rab proteins switch between a cytosolic, GDP-bound inactive state and a membrane-anchored, GTP-bound active state. By themselves, Rabs show slow rates of GDP/GTP exchange and GTP hydrolysis. Thus, Rab proteins are considered...
01:25Rab Cascades
Rab GTPases act in a regulated cascade during membrane fusion, helping the lipid bilayers mix. The Rab family of proteins are active when bound to GTP, and inactive when bound to GDP. Hence, they act as guanine nucleotide-dependent molecular switches. Rab-GTP recognizes and binds to long or short-range tethering proteins to capture the target vesicle. These tethers coordinate with SNAREs on the vesicle and the target membrane to assemble the trans SNARE complex that locks the mixing bilayers.