The glutathione S‐transferase (GST) pull‐down assay was performed according to the previously described methods (Mehto et al, 2019a (link), 2019b ). Briefly, GST or GST‐RIPK2 or GST‐IRGM recombinant proteins were expressed in E. coli SoluBL21 (Amsbio), and the proteins were purified on Glutathione Sepharose 4 Fast‐Flow beads (GE Healthcare). The [35S] labeled‐ Myc‐NOD1, Myc‐NOD2, Myc‐RIPK2, Myc‐ULK1, Myc‐ATG16L1 or Myc‐Beclin‐1 proteins were in vitro translated using TnT T7–coupled reticulocyte lysate system (Promega). The GST proteins were incubated with [35S]‐labeled proteins in 200 μl of NETN‐E buffer (50 mmol/l Tris, pH 8.0, 100 mm NaCl, 6 mm EDTA, 0.5% NP‐40, and 1 mm dithiothreitol (DTT) supplemented with complete mini EDTA‐free protease inhibitor cocktail; Roche) for 2 h at 4°C. After incubation, the beads were washed five times with NETN‐E buffer, boiled with loading buffer, and subjected to SDS–PAGE. The gels were stained with coomassie blue and vacuum‐dried. The GST was detected by staining with coomassie blue stain, whereas the [35S]‐ labeled Myc‐tagged proteins were detected in PharosFX imager (Bio‐Rad Laboratories).
E coli solubl21
Manufactured by AMS Biotechnology
E. coli SoluBL21 is a strain of Escherichia coli designed for the efficient expression of recombinant proteins. It is a chemically competent cell line optimized for improved solubility of expressed proteins.
Automatically generated - may contain errors
Lab products found in correlation
Glutathione sepharose 4 fast flow beads, by GE Healthcare (1 mentions)
Pharosfx imager, by Bio-Rad (1 mentions)
Complete mini edta free protease inhibitor cocktail, by Roche (1 mentions)
Tnt t7 coupled reticulocyte lysate system, by Promega (1 mentions)
Hitrap q hp, by GE Healthcare (1 mentions)
Ni nta agarose bead, by Qiagen (1 mentions)
2 protocols using e coli solubl21
1
GST Pull-Down Assay for Protein Interactions
2
Recombinant Ubiquitin Protein Production
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pET-His-Ub G76C was a generous gift from T. Yao (23 (link)). Ub plasmid DNA was transformed into E. coli SoluBL21 (Amsbio, catalog no. C700200) competent cells and grown in 2xYT-Amp medium. Ub G76C (Ub G76C) was expressed as soluble protein by inducing with 0.4 mM IPTG for 4 hours at 37°C upon the culture reaching optical density at 600 nm = 0.4 to 0.6. Bacteria cells were harvested and lysed (AvestinEmulsiflexC3). Protein was purified through Ni-NTA agarose beads (Qiagen) [lysis buffer: 300 mM NaCl, 50 mM tris (pH 8.0), 10 mM imidazole, 5 mM 2-mercaptoethanol (BME), 1x protease inhibitor; elution buffer: 300 mM NaCl, 50 mM tris (pH 8.0), 300 mM imidazole, and 5 mM BME] followed by HiTrap Q HP (GE Healthcare) liquid chromatography column [buffer A: 50 mM NaCl, 20 mM tris (pH 8.0), 0.2 mM EDTA, and 10 mM BME; buffer B: 1 M NaCl, 20 mM tris (pH 8.0), 0.2 mM EDTA, and 10 mM BME]. Purified Ub was then dialyzed against water supplemented with 1 mM acetic acid followed by flash freezing in liquid nitrogen and lyophilized using a Sentry lyophilizer (VirTis).
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