Use of a native affinity ligand for the detection of G proteins by capillary isoelectric focusing with laser-induced fluorescence detection.
Affinity probe capillary isoelectric focusing (CIEF) with laser-induced fluorescence was explored for detection of Ras-like G proteins. In the assay, a fluorescent BODIPY FL GTP analogue (BGTPgammaS) and G protein were incubated resulting in formation of BGTPgammaS-G protein complex. Excess BGTPgammaS was separated from BGTPgammaS-G protein complex by CIEF using a 3-10 pH gradient and detected in whole-column imaging mode. In other cases, a single point detector was used to detect zones during the focusing step of CIEF using a 2.5-5 pH gradient. In this case, analyte peaks passed the detector in approximately 5 min at an electric field of 350 V/cm. Detection during focusing allowed for more reproducible assays at shorter times but with a sacrifice in sensitivity compared to detection during mobilization. Resolution was adequate to separate BGTPgammaS-Ras and BGTPgammaS-Rab3A complexes. Formation of specific complexes was confirmed by adding GTPgammaS to samples containing BGTPgammaS-G protein. GTPgammaS competed with BGTPgammaS for G protein binding sites resulting in decreased BGTPgammaS-G protein peak heights. The concentrating effect of CIEF enabled detection limits of 30 pM.
Cunliffe
Jennifer M
JM
Department of Chemistry, niversity of Michigan, Ann Arbor, MI 48109-1055, USA.
Liu
Zhen
Z
Pawliszyn
Janusz
J
Kennedy
Robert T
RT
D000818:Animals; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D019204:GTP-Binding Proteins / Q000737:chemistry*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000737:chemistry*; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods*; D007834:Lasers; D008024:Ligands
D005456:Fluorescent Dyes; D008024:Ligands; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins