Figures & data
Figure 1. Comparison of LC/MS chromatograms of Abl1 reaction solutions with different volumes of extract solution of BT: (a) 0 µL; (b) 126 µL; (c) 63 µL; (d) 25.2 µL. LC separation was performed on a Symmetry® C-18 column (150 × 2.1 mm i.d., particle size 5 µm) (Waters, Milford, MA) at room temperature (23 °C) with a flow rate of 0.2 mL/min. Mobile phase A (mpA) was 0.1% trifluoroacetic acid (TFA) in 25% acetonitrile; mobile phase B (mpB) was water. Gradient program: 0–2 min, 69% of mpA (isocratic); 2–6 min, 69 to 80% of mpA (linear gradient); 6–10 min, 69% of mpA (isocratic). A: peak area of p-Abltide; P: p-Abltide; S: Abltide.
Figure 2. Comparison of LC-UV chromatograms of Abl1 reaction solutions with different volumes of extract solution of eppendorf plastic tubes: 4 (0 µL); 3 (25.2 µL); 2 (63 µL); 1 (126 µL). Analysis was carried out at 210 nm with an Alltima C18 5 µm, 250 mm × 4.6 mm column (Alltech Associates, Lokeren, Belgium). The temperature of the column was kept at 30 °C, while the temperature of the sampler chamber was set to 4 °C. The injection volume was 10 µL. Mobile phase was prepared by combining water, acetonitrile and TFA in a 78:22:0.05 (v/v/v) ratio. The mobile phases were degassed by sparging with helium for 2 min. The flow rate was set to 0.5 mL/min. p-Abltide is shown in the dashed rectangle.
Figure 3. LC-UV chromatograms of microcentrifuge tubes extract with λ = 210 nm (a) and λ = 254 nm (b). Trace 1 represents freshly prepared blank of reaction buffer, trace 2 is GTE and trace 3 BTE. DTT1 and DTT2 represent the reduced and oxidized forms of DTT, respectively. Due to air oxidation, the reduced form of DTT was converted to the oxidized form. The time from preparation to injection was different for the blank solution and the tube extraction solutions, which caused one extra peak (DTT1) in the chromatogram of trace 1. LC separation conditions were identical to those mentioned in , except the flow rate was 0.2 mL/min, which is comparable to the LC/MS method.
