The bait proteins and target proteins were respectively cloned into pGEX-4T-1 and pET-32a to express GST- and His-fusion proteins in E. coli strain BL21 Gold (DE3). The fusion proteins were expressed and purified by the method [36] (link). The proteins were induced by adding 0.5 mM isopropylthio-β-Dgalactopyranoside (IPTG, Sigma) at 16 ℃ for 16 h, and the GST- fusion proteins were purified using a glutathione (GST) agarose affinity chromatography column (Glutathione Sepharose 4B, GE Healthcare, Sweden), while His-fusion proteins were purified using a histidine-labeled affinity chromatography column (Ni Sepharose HP, GE Healthcare, Sweden). GST-mediated pulldown assay was performed as described by the method [36] (link). The GST- fusion proteins were separately mixed with His-fusion proteins by 3:1 and then incubated with the prewashed glutathione (GST) beads at 4 ℃ overnight. The pGEX-4T-1 expressed with His-fusion proteins were used as the negative control. The bound proteins were eluted and mixed with SDS loading buffer, boiled, and then analyzed using SDS-PAGE and immunoblotting with a GST-Tag Mouse mAb (ABclonal, Wuhan, China) and a His-Tag Mouse mAb (ABclonal, Wuhan, China).
Isopropylthio β dgalactopyranoside iptg
Manufactured by Merck Group
Sourced in Sweden, Canada
Isopropylthio-β-D-galactopyranoside (IPTG) is a synthetic chemical compound used as a molecular biology tool in the laboratory. Its core function is to serve as an inducer, triggering the expression of genes under the control of the lac operon in bacterial cells.
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Lab products found in correlation
2 protocols using isopropylthio β dgalactopyranoside iptg
1
Bait-Target Protein Interaction Assay
2
Genomic DNA Purification and Bacterial Culture
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The Wizard® genomic DNA purification kit used was from Promega (Madison, WI, USA). The oligonucleotides used in this work (listed in Supplementary data, Table S1) were Hammerstein casein was provided by Merck (Darmstadt, Germany). Kanamycin sulfate and ampicillin sodium salt USP were purchased from Bio Basic (Markham, Canada Inc.).
Isopropyl-thio-β-D-galactopyranoside (IPTG) and lysozyme were bought from Sigma-Aldrich Co (St Louis, MO, USA).
The culture media used were Luria-Bertani (LB) and yeast extract peptone dextrose (YPD), buffered glycerol-complex (BMGY), and buffered methanol-complex (BMMY). The YPD media were composed of yeast extract, 10 g; peptone, 20 g; and dextrose, 20 g per liter.
The LB and YPD were complemented with 20 g/L agar to make solid media. The BMGY/BMMY medium was composed of yeast extract, 1%; peptone, 2%; biotin, 4×10 -5 % (w/v); and yeast nitrogen base, 1.34% in 100 mM potassium phosphate pH 6 plus glycerol 1%
Isopropyl-thio-β-D-galactopyranoside (IPTG) and lysozyme were bought from Sigma-Aldrich Co (St Louis, MO, USA).
The culture media used were Luria-Bertani (LB) and yeast extract peptone dextrose (YPD), buffered glycerol-complex (BMGY), and buffered methanol-complex (BMMY). The YPD media were composed of yeast extract, 10 g; peptone, 20 g; and dextrose, 20 g per liter.
The LB and YPD were complemented with 20 g/L agar to make solid media. The BMGY/BMMY medium was composed of yeast extract, 1%; peptone, 2%; biotin, 4×10 -5 % (w/v); and yeast nitrogen base, 1.34% in 100 mM potassium phosphate pH 6 plus glycerol 1%
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