Gst protein interaction pull down kit

The expression vectors with either GST-labeled NEDD4L or His-labeled SP1 were constructed by Shanghai Genechem Co., Ltd. GST Spin Purification Kit (16107) and GST Protein Interaction Pull-Down Kit (21516) were purchased from Thermo.
Protein purification: briefly, centrifuging column at 700 × g for 2 min to remove storage buffer and equilibrate column with two resin-bed volumes of equilibration buffer. Add the prepared protein extract to the column and allow it to enter the resin bed. Then centrifuging column at 700 × g for 2 min and collect the flow-through in a centrifuge tube. Washing resin with two resin-bed volumes of equilibration buffer. Centrifuging at 700 × g for 2 min and collect fraction in a centrifuge tube. Eluting GST-tagged protein from the resin by adding one resin-bed volume of elution buffer. Protein pull-down assay: briefly, immobilizing the obtained GST-NEDD4L protein on the glutathione agarose according to the instructions. Adding in prepared prey protein sample and incubated at 4 °C for at least 1 h. Centrifuging at 1250 × g for 30 s to 1 min. Adding in 400µL of wash solution and repeat washing for a total of five washes. Eluting with glutathione elution buffer and collecting eluent for analysis.

The ORF of Clg2p was amplified with primers 13F/R and inserted into pGEX-6P-1 to create a GST-Clg2p bait protein. The ORFs of Clf and ClUrase were amplified using primers 14F/R and 15F/R, respectively, and inserted into pET-28a vectors to create a His-Clf/ClUrase prey protein. The BL21 (DE3) strain was used to express the protein. In vitro pull-down assays were performed following the manufacturer’s instructions (Pierce™ GST Protein Interaction Pull-Down Kit (#21516, http://www.thermoscientific.com)). The bait and prey were eluted from 25 μL glutathione agarose. Pull-down proteins were then resolved by 12% SDS-PAGE and detected by Western blotting. GST-Clg2p and HIS-Clf/His-ClUrase fusion proteins were detected by immunoblotting with 1:2,000 diluted anti-GST antibody (#10000-0-AP, PROTEINTECH, http://www.ptgcn.com) and 1:1,500 diluted anti-His (#10001-0-AP, PROTEINTECH) antibody (Millipore), respectively. The goat anti-rabbit IgG (peroxidase conjugate) secondary antibody (Boster, http://www.boster.com.cn) was detected using DBA Western Blotting Substrate (Boster, http://www.boster.com.cn) following the manufacturer’s protocol.

The indicated FIP200 and RIG-I domains were cloned into pGEX-5X-3 (GE Healthcare, # 28-9545-55) to fuse with a GST tag, pET28b(+) (Novagen, # 69865-3) to fuse with a His tag, pT7-FLAG-2 (Sigma, # P1243) for a FLAG tag, or pMXB10 (New England Biolabs, # E6901S) for a MBP tag. These constructs were transfected into BL21 (DE3) E. coli (New England Biolabs, # C2527I) and cultured in LB broth at 20 °C. IPTG (0.4 mM) was added to induce protein expression. MBP-tagged proteins were purified using the IMPACT kit (New England Biolabs, # E6901S), and the MBP pull-down assays were performed using the anti-MBP Magnetic Beads New England Biolabs, # E8037S). FLAG-tagged proteins were purified by using the EZview Red Anti-FLAG M2 Affinity Gel (Sigma, # F2426). The GST Protein Interaction Pull-Down Kit (ThermoFisher Scientific, # PI21516) was used for GST-tagged protein purification and GST pull-down assays. The His-Spin Protein Miniprep kit (Zymo Research, # P2002) was used for His-tagged protein purification and His pull-down assays.