Pierce gst spin purification kit

To obtain recombinant protein, the MdCML15 cDNA was introduced into the pET32a vector, while the MdBT2 cDNA was integrated into the pGEX-4 T-1 vector. These constructed plasmids, along with the pGEX-4 T-1 vector itself, were introduced into Escherichia coli BL21 (DE3) for the expression of HIS–MdCML15, GST–MdBT2, and GST proteins, respectively. In the pull-down assay, 6 μg of HIS–MdCML15 was incubated with 10 μg of either GST–MdBT2 or GST. The pull-down procedure was performed using the Pierce GST Spin Purification Kit (Thermo).

CoIP and Western blot were performed as described in the previous study [43 (link)]. GST-SNAI1 plasmid was constructed with pGEX-4T-2, the GST-SNAI1 purification was carried out as described previously [58 (link)]. The GST-pulldown was performed using Pierce® GST Spin Purification Kit (ThermoFisher, 16106) according to the manufacture instruction. Cells were lysed with the RIPA lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate and protease inhibitor cocktail (Biomake)). After centrifugation at 12, 000 g for 15 min at 4 °C, the supernatant was incubated with the indicated antibody or with control IgG and Protein A/G at 4 °C for 4 h. After centrifugation at 3,000 g for 10 min at 4 °C, the supernatant was abandoned and the precipitate was subjected to wash three times with washing buffer. Then the pellets were suspended with protein 2 × loading buffer, boiling at 100 °C for 5 min and subjected to SDS-PAGE. After electrophoresis, proteins were separated and blotted onto a PVDF membrane (Millipore). Membranes were probed with the specific primary antibody and then peroxidase-conjugated secondary antibodies. The bands were visualized by chemiluminescence.

To achieve high expression levels of the target proteins (the cytoplasmic domains of OrhK-GST and His₆-OrhR), recombinant plasmids were transformed into competent E. coli BL21 cells, and their expression was induced by IPTG under optimal growth conditions. The cells were harvested by centrifugation, washed once with lysis buffer, and stored at -80°C until use. The frozen cells were resuspended in lysis buffer containing phenylmethylsulfonyl fluoride and lysozyme and further lysed by sonication. After centrifugation, the supernatant was extracted with a nickel-nitrilotriacetic acid-agarose suspension (Qiagen) or a Pierce GST Spin Purification Kit (Thermo) as described by the manufacturer. Proteins were eluted, recovered and dialyzed against storage buffer [54 (link)]. Finally, the protein concentrations were determined with a protein assay kit (Bio-Rad), and the purity was assessed by SDS-PAGE.

The LlWRKY22 ORF was cloned into the pGEX-4 T-1 vector to generate GST-LlWRKY22 fusion proteins. The GST-LlWRKY22 proteins were synthesized in Escherichia coli BL21 cells via the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG). The accumulation of GST-LlWRKY22 proteins was detected by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The GST-LlWRKY22 and GST proteins were enriched using a Pierce GST Spin Purification Kit (Thermo Fisher, New York, NY, USA). The EMSA probes were labeled with biotin at the 5′-end and the EMSA was performed with an EMSA kit (Thermo Fisher).

The GST alone and GST fusion protein were expressed in E. coli BL21 (Takara), and purified by Pierce GST Spin Purification Kit (Thermo scientific). GST pull-down assay was performed using a Pierce GST Protein Interaction Pull-Down Kit (Thermo scientific). The purified GST-tagged fusion protein (BAIT) was immobilized on the Pierce Spin Column. MCF-7 cells were lysed in pull-down lysis buffer containing DNase (Takara). The supernatant was loaded onto the Pierce Spin Column, and then incubated at 4°C for 2 hour with gentle agitation. The column was centrifuged at 1250 × g for 1 minute and the flow through was discarded. Then the column was washed five times using wash solution. Elution buffer was added to the column followed by 5-min incubation with gentle agitation. After that, the column was centrifuged at 1250 × g for 1 minute, and the eluent was subjected to Western blot assay.