FIGURE 4.
Unlabeled binding partners compete with Gαi1-Tb for binding to CrV2-Alexa. TR-FRET measurements were taken with the following parameters: λex 340 nm, λem 572 nm, 50-μs delay, and 900-μs counting duration. A, unlabeled Gαi1 competes for binding to CrV2-Alexa reducing the TR-FRET signal. B, unlabeled CrV2 competes for binding to Gαi1-Tb reducing the TR-FRET signal. A and B, 10 nm Gαi1-Tb was mixed with 20 nm CrV2-Alexa in 100 μl of TMN buffer. At 5 min, 2 μm of unlabeled Gαi1 or 70 nm of unlabeled CrV2 (■) or an equivalent volume of TMN buffer (▴) was added and TR-FRET measurements over the time period shown. Backgrounds of 10 nm Gαi1-Tb with buffer added at 5 min (●) and 10 nm Gαi1-Tb + the indicated unlabeled protein (♦) are shown. Data shown are mean (n = 2). C, Gβ4γ2 inhibits CrV2-Alexa association with Gαi1-Tb. 10 nm Gαi1-Tb was mixed with 20 nm CrV2-Alexa with or without 240 nm Gβ4γ2 in 100 μl of TMN buffer. Backgrounds of Gαi1-Tb and Gαi1-Tb + Gβ4γ2 have been deducted as appropriate. Data shown are mean (n = 2). a.u., arbitrary units.