Thermal shift assays were performed with a StepOnePlus real-time polymerase chain reaction (PCR) machine (Life Technologies) using SYPRO Orange dye (Invitrogen) and thermal ramping (0.3°C in step intervals between 25° and 94°C). All proteins were diluted to a final concentration of 5 μM in 50 mM tris-HCl (pH 7.4) and 100 mM NaCl in the presence or absence of the indicated concentrations of ATP, H2O2, DTT, GSH, or MLN8237 [final dimethyl sulfoxide (DMSO) concentration no higher than 4% (v/v)] (131) and were assayed as described previously (67). Normalized data were processed using the Boltzmann equation to generate sigmoidal denaturation curves, and average Tm/∆Tm values were calculated as previously described (132) using GraphPad Prism software.

注意:以上内容是从某篇研究文章中自动提取的,可能无法正确显示。



Q&A
请登录并在线提交您的问题
您的问题将发布在Bio-101网站上。我们会将您的问题发送给本研究方案的作者和具有相关研究经验的Bio-protocol成员。我们将通过您的Bio-protocol帐户绑定邮箱进行消息通知。