His-STING (139aa–379aa) was expressed and purified as previously described (Tanaka and Chen, 2012 (link)). Full-length GST, GST-STX17, FLAG-STX17, FLAG-VAMP8, and FLAG-SNAP29 were expressed individually with amino-terminal TEV protease cleavable GST tag in Escherichia coli strain BL21(DE3) at 25°C. All proteins were purified according to previously described protocols (Diao et al., 2015 (link)). In GST pull-down assays, GST and GST-STX17 were applied to the GST resin. Then 1 µg SNAP29 and 1 µg STING-His (139aa–379aa) were added respectively and incubated overnight. Proteins were eluted by reduced glutathione and dissolved in sample buffer for SDS–PAGE and immunoblotting. In His pull-down assay, 1 µg FLAG-tagged proteins and 1 μg His-tagged STING (139aa–379aa) were incubated overnight with Nickel NTA resin (Qiagen) and then dissolved in sample buffer for SDS–PAGE and immunoblotting.
Protocol detail
STING Interaction Mapping Using Pull-Down Assays
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Assay
Buffer
Escherichia coli
Nickel
Proteins
Reduced glutathione
Resin
Sds page
Snap29
Strain
Stx17
Tev protease
Vamp8
Corresponding Organization : The University of Texas Southwestern Medical Center
Other organizations : Huazhong University of Science and Technology, Shanghai Jiao Tong University, Howard Hughes Medical Institute, Renji Hospital
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Variable analysis
independent variables
- Expression and purification of His-STING (139aa–379aa)
- Expression and purification of full-length GST, GST-STX17, FLAG-STX17, FLAG-VAMP8, and FLAG-SNAP29 in Escherichia coli strain BL21(DE3) at 25°C
- Interactions between STING-His (139aa–379aa) and GST-STX17, FLAG-STX17, FLAG-VAMP8, and FLAG-SNAP29 in GST and His pull-down assays
- Expression and purification of GST as a control in GST pull-down assays
- Incubation of 1 µg SNAP29 and 1 µg STING-His (139aa–379aa) with GST and GST-STX17 in GST pull-down assays
- Incubation of 1 µg FLAG-tagged proteins and 1 μg His-tagged STING (139aa–379aa) with Nickel NTA resin in His pull-down assays
- GST as a control in GST pull-down assays
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