Pierce gst protein interaction pull down kit

We used previously purified protein LONP1‐GST synthesized by Gene Universal (Anhui, China) as the bait protein, and the prey protein HMGCS2 was from 293T cell lysates. The experimental procedure was carried out according to the Pierce™ GST Protein Interaction Pull‐Down Kit (ThermoFisher, cat: 21516).

DNA for the BRCT domains of human BARD1 was fused with a GST tag and the construct subcloned into the bacterial expression vector, pETBlue-1 with NovaBlue Singles Competent Cells (Millipore-Sigma). The expression constructs were then transformed into Tuner (DE3)pLacI (Millipore-Sigma), a derivation of BL21 strain, for inducible overexpression. Fusion proteins were induced with a standard IPTG method for 3 h and crude bacterial lysate was prepared according to manufacturer’s protocol. The GST pulldown assay was performed with Pierce GST Protein Interaction Pull-Down Kit (ThermoFisher Scientific) using GST-BRCT and either WT or mutant 6His-p50 as indicated. GST fusion proteins were immobilized on glutathione-Sepharose beads and after washing the interacting complex was eluted and analyzed on SDS–PAGE followed by immunoblotting.

GST-tagged ORF expression clone for LGALS1 in bacteria was purchased from Genecopoeia (EX-I0309-B04). To verify the expression of Gal-1 protein expression, we transformed this plasmid in BL21 (DE3) E. coli competent cells. Fusion protein expression was induced by adding 0.5 mM IPTG for three hours. Bacteria were lysed with 2 mg/ml lysozyme in a lysis buffer. Glutathione agarose was equilibrated in Pierce spin columns using wash buffer, followed by the addition of 200 µg of the GST fusion protein for immobilization. The spin column was placed in a gentle rocking motion on a rotating platform for 3 h incubated at 4 °C. Then, the spin column tube with the mixture was centrifuged and washed five times with a wash solution. To the bound fraction, we added 200 µg of total protein of GSC33 control cells. The GST pulldown assay was performed from the Pierce GST Protein Interaction Pull-Down Kit (Thermo Scientific) using the manufacturer’s instructions.

A Pierce GST Protein Interaction Pull-Down Kit purchased from Thermo Scientific (cat. No. 21516) and recombinant proteins including a human USP8-6*His fusion protein (Ag27104, Proteintech), a human CD274-GST fusion protein (Ag12432, Proteintech), and recombinant human GST (ab70456, Abcam), were used to carry out the GST pull-down assay following the producer’s protocol.

His-tagged gp70.1, GST-tagged FlgM, GST-tagged RpoS and GST alone were purified with Ni-agarose or glutathione-agarose. GST pull-down assays were performed by Pierce GST Protein Interaction Pull-Down kit (Thermo Scientific, USA) according to the manufacturer’s instructions. Samples were separated by SDS-PAGE, stained with Coomassie blue, and the captured His-gp70.1 was detected by a monoclonal antibody against the His tag.

GST-FHC was expressed in E Escherichia coli BL21 (DE3) cells and GFP-NS4B was generated in HEK293T cells. All procedures were performed according to the Pierce® GST Protein Interaction Pull-Down Kit manufacturer’s instructions (Thermo, USA). In brief, GST or GST-FHC expressed in E. coli BL21 cells was treated with pull-down lysis buffer on ice for 30 min, followed by immobilization on an equilibrated glutathione agarose resin for 2 h at 4 °C. The resin was then washed five times with wash solution (TBS: pull-down lysis buffer = 1:1). HEK293T cell cultures were treated as described above. Supernatants were added to the resin and incubated overnight at 4 °C. After washing five times, proteins were eluted with glutathione elution buffer. The eluted proteins were detected by Western blotting with anti-GFP antibody. In addition, the interaction between GST-NS4B and GFP-FHC was also verified as described above.

For GST pull-down assays, GST or GST-ORF5 protein was expressed in Escherichia coli Rosetta (DE3) cells and Flag-GPNMB protein was expressed in HEK293T cells. The Pierce GST Protein Interaction Pull-Down Kit (Thermo Fisher Scientific, United States) was used according to the manufacturer’s instructions. The proteins produced in E. coli were treated with pull-down lysis buffer and then conjugated to glutathione beads glutathione agarose resin for 2 h at 4°C. The beads then were washed with 1:1 wash solution (TBS: Pull-Down Lysis Buffer) for five times and incubated with Flag-GPNMB harvested from HEK293T cells overnight at 4°C. After washing five times, target protein was detected by western blot.