Chinese Bulletin of Botany ›› 2016, Vol. 51 ›› Issue (6): 790-800.DOI: 10.11983/CBB15226
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Ruixue Yang†, Haiyang Liu†, Shengli Liu, Tingqiao Yu, Yuzhen Chen*, Cunfu Lu*
Received:
2015-12-30
Accepted:
2016-03-21
Online:
2016-11-01
Published:
2016-12-02
Contact:
Chen Yuzhen,Lu Cunfu
About author:
# Co-first authors
Ruixue Yang, Haiyang Liu, Shengli Liu, Tingqiao Yu, Yuzhen Chen, Cunfu Lu. Optimization of Detection Methods for Zea mays ABA Receptor ZmPYL1 by Isothermal Titration Calorimetry[J]. Chinese Bulletin of Botany, 2016, 51(6): 790-800.
Figure 2 Ultrasonic treatments for bacterial resuspension of different concentrations (A) A1-A4 indicate the comparison of 200, 100, 70 and 40 mg·mL-1 bacterial suspension before (left beaker) and after (right beaker) ultrasonic treatments; (B) Effect of bacterial resuspension of different concentrations on the OD600 before (0 min) and after (15 min) ultrasonic treatments; (C) Effect of bacterial resuspension of different concentrations on the protein content before (0 min) and after (15 min) ultrasonic treatments; (D) SDS-PAGE detection of protein content in sediment and supernatant of different bacterial concentration, lane 1, 3, 5, 7 represent the protein content in sediment of 200, 100, 70 and 40 mg·mL-1 bacterial suspension, respectively; and lane 2, 4, 6, 8 represent the protein content in supernatant of 200, 100, 70 and 40 mg·mL-1 bacterial suspension, respectively. Target protein band is showed by arrow.
Figure 3 Ultrasonic treatment of inclusion body ZmPYL4 (A) Effect of total ultrasonic time on the OD600 of bacterial resuspension and the protein content in supernatant; (B) Bacterial resuspension before (left beaker) and after (right beaker) ultrasonic treatment for 30 min; (C) SDS-PAGE detection of protein level in sediment and supernatant of bacterial suspension. Lane 1: The protein level in sediment before ultrasonic treatment. Lane 2: The protein level in supernatant before ultrasonic treatment. Lane 3: The protein level in sediment after ultrasonic treatment for 30 min. Lane 4: The protein level in supernatant after ultrasonic treatment for 30 min.
Figure 6 SDS-PAGE detection of purified ZmPYL1 protein Lane 1: The protein level in sediment after ultrasonic treatment; Lane 2: The protein level in supernatant after ultrasonic treatment; Lane 3: The collecting liquid of the supernatant through Ni-NTA; Lane 4: The collecting liquid of wash buffer through Ni-NTA; Lane 5-8: The collecting liquid of elution buffer through Ni-NTA for the first, second, third and fourth time, respectively.
Thermodynamic parameters | 2 mmol·L-1 (±)-ABA+ 0.005 mmol·L-1 ZmPYL1 | 2 mmol·L-1 (±)-ABA+ 0.1 mmol·L-1 ZmPYL1 | 4 mmol·L-1 (±)-ABA+ 0.1 mmol·L-1 ZmPYL1 |
---|---|---|---|
N (sites) | 15.5±5.52 | 0.651±0.190 | 0.821±0.069 |
Kd (μmol·L-1) | 135.32 | 72.99 | 72.46 |
Table 1 The thermodynamic parameters of (±)-ABA and ZmPYL1 protein in different concentrations
Thermodynamic parameters | 2 mmol·L-1 (±)-ABA+ 0.005 mmol·L-1 ZmPYL1 | 2 mmol·L-1 (±)-ABA+ 0.1 mmol·L-1 ZmPYL1 | 4 mmol·L-1 (±)-ABA+ 0.1 mmol·L-1 ZmPYL1 |
---|---|---|---|
N (sites) | 15.5±5.52 | 0.651±0.190 | 0.821±0.069 |
Kd (μmol·L-1) | 135.32 | 72.99 | 72.46 |
Figure 7 The variation of differential power with time and the heat variation of injection with the molar ratio(A) 2 mmol·L-1 (±)-ABA was titrated into 0.05 mmol·L-1 ZmPYL1; (B) 2 mmol·L-1 (±)-ABA was titrated into 0.1 mmol·L-1 ZmPYL1; (C) 4 mmol·L-1 (±)-ABA was titrated into 0.1 mmol·L-1 ZmPYL1; (D) 4 mmol·L-1 (±)-ABA was titrated into HEPES solution without protein. The solid lines in bottom panels represent the fitting curves, and the closer the fitting curve is to experimental dots, the more credible the fitting result is.
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