Detection of G proteins by affinity probe capillary electrophoresis using a fluorescently labeled GTP analogue.
An affinity probe capillary electrophoresis (APCE) assay for guanine-nucleotide-binding proteins (G proteins) was developed using BODIPY FL GTPgammaS (BGTPgammaS), a fluorescently labeled GTP analogue, as the affinity probe. In the assay, BGTPgammaS was incubated with samples containing G proteins and the resulting mixtures of BGTPgammaS-G protein complexes and free BGTPgammaS were separated by capillary electrophoresis and detected with laser-induced fluorescence detection. Separations were completed in less than 30 s using 25 mM Tris, 192 mM glycine at pH 8.5 as the electrophoresis buffer and applying 555 V/cm over a 4-cm separation distance. BGTPgammaS-Galpha(o) peak heights increased linearly with Galpha(o) up to approximately 200 nM using a 50 nM BGTPgammaS probe. The detection limit for Galpha(o) was 2 nM, corresponding to a mass detection limit of 3 amol. The high speed of the APCE assays allowed reaction kinetics and the dissociation constant (Kd) to be determined. The on-rate and off-rate of BGTPgammaS to Galpha(o) were 0.0068 +/- 0.0004 and 0.000 23 +/- 0.000 01 s(-1), respectively. The half-life of the BGTPgammaS-Galpha(o) complex was 3060 +/- 240 s and Kd was 8.6 +/- 0.7 nM. The estimates of these parameters are in good agreement with those obtained using established techniques, indicating the suitability of this method for such measurements. Lowering the temperature of the separation improved the detection of the complex, allowing the assay to be performed on a commercial instrument with longer separation times. Additionally, the capability of the technique to detect several G proteins based on their binding to BGTPgammaS was demonstrated with assays for Galpha and Galpha(i1) and for Ras and Rab3A.
Jameson
Emily E
EE
Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
Cunliffe
Jennifer M
JM
Neubig
Richard R
RR
Sunahara
Roger K
RK
Kennedy
Robert T
RT
D000818:Animals; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes; D044385:GTP-Binding Protein alpha Subunits / Q000032:analysis; D019204:GTP-Binding Proteins / Q000032:analysis*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D007700:Kinetics; D016283:Proto-Oncogene Proteins p21(ras) / Q000032:analysis; D013696:Temperature
C486958:BODIPY FL GTPgammaS; D005456:Fluorescent Dyes; D044385:GTP-Binding Protein alpha Subunits; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D016283:Proto-Oncogene Proteins p21(ras)