Bacterial Expression and In Vitro Interaction

GST fusion proteins were expressed in BL21 (Vazyme) Escherichia coli, followed by ultrasonic disruption and purification using glutathione Sepharose 4B beads (GE). In vitro transcription and translation experiments were performed using rabbit reticulocyte lysate (TNT systems, Promega) according to the manufacturer’s recommendation. The beads were co-incubated with the in vitro translated proteins and then washed with binding buffer. The eluates were then analyzed using SDS-PAGE.

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Zhang D.G., Yu W.Q., Liu J.H., Kong L.G., Zhang N., Song Y.D., Li X.F., Fan Z.M., Lyu Y.F., Li N, & Wang H.B. (2023). Serum/glucocorticoid-inducible kinase 1 deficiency induces NLRP3 inflammasome activation and autoinflammation of macrophages in a murine endolymphatic hydrops model. Nature Communications, 14, 1249.

Publication 2023

glutathione Sepharose 4B beads TNT systems

Buffer Escherichia coli Glutathione Promega Proteins Rabbit Reticulocyte Sds page Sepharose 4b Transcription Ultrasonic

Corresponding Organization :

Other organizations : Shandong Provincial Hospital, Shandong University

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Variable analysis

independent variables

Expression of GST fusion proteins in BL21 (Vazyme) Escherichia coli
Purification of GST fusion proteins using glutathione Sepharose 4B beads (GE)

dependent variables

Binding of in vitro translated proteins to the GST fusion proteins bound to the glutathione Sepharose 4B beads
Elution of bound proteins from the beads

control variables

Ultrasonic disruption of Escherichia coli cells expressing the GST fusion proteins
In vitro transcription and translation of proteins using rabbit reticulocyte lysate (TNT systems, Promega)
Washing of the beads with binding buffer

controls

Positive control: Not specified
Negative control: Not specified

Annotations

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