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Related Concept Videos

GTPases and their Regulation02:14

GTPases and their Regulation

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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,...
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Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and GTPase-linked Immunosorbent Assay
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Active GTPase Pulldown Protocol.

Martin J Baker1, Ignacio Rubio2,3

  • 1Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Methods in Molecular Biology (Clifton, N.J.)
|May 12, 2021
PubMed
Summary
This summary is machine-generated.

This study presents a label-free method to measure the activation state of Ras GTPases, crucial proteins involved in cell signaling and diseases like cancer. The technique isolates active, GTP-bound Ras for analysis, aiding research into cellular functions and disease mechanisms.

Keywords:
ActivationArfCdc42GTPasePulldownRBDRabRacRalRanRapRasRhoRin/Rit

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cellular Signaling

Background:

  • Ras and related small GTPases are key regulators of cellular functions.
  • Dysregulation of these GTPases is linked to diseases, including cancer and Rasopathies.
  • Understanding GTPase activation is vital for studying signal transduction.

Purpose of the Study:

  • To describe a broadly employed, label-free method for determining Ras GTPase activation state.
  • To enable qualitative and semi-quantitative analysis of active Ras and related GTPases.
  • To facilitate the investigation of small GTPase biochemical functions.

Main Methods:

  • Affinity-based isolation of the active, GTP-bound fraction of Ras from cellular extracts.
  • Production of recombinant affinity probes (baits) that bind active GTPases.
  • Pulldown assay for isolating active GTPase fractions from various cell types, followed by western blotting.

Main Results:

  • The described method allows for rapid, label-free determination of Ras GTPase activation.
  • The protocol is adaptable for analyzing other related GTPases.
  • Reproducible measurement of active Ras or Ras family GTPases is achievable in diverse cellular contexts.

Conclusions:

  • This method provides a robust approach for assessing Ras GTPase activation.
  • It is valuable for research into the roles of small GTPases in normal physiology and disease.
  • The technique supports the investigation of cellular signaling pathways involving Ras family proteins.