BL21 E. coli strains (RBC Bioscience, Taipei, Taiwan) were transformed with the pET-DEST42-Rho-OR2AT4-6xHis plasmid and incubated in Luria-Bertani (LB) broth (BD Difco, Sparks, MD, USA) containing 100 μg/mL ampicillin. The cells were grown at 37 °C with shaking at 200 rpm until the optical density at 600 nm reached to 0.6–0.8. Protein expression was induced with 1 mM IPTG at 16 °C for 16 h at 200 rpm. Cells were harvested at 6000×g for 10 min and lysed by sonication for 20 min (5 s on/off). Homogenized cells were centrifuged (12,000×g, 30 min) and separated into soluble/insoluble fractions. Insoluble fraction was solubilized in solubilization buffer (0.1 M Tris-HCl, 20 mM SDS, 1 mM EDTA, and 100 mM DTT) and dialysis by 10 kDa MWCO Slide-A-Lyzer Dialysis Cassette (Thermo Fisher Scientific, Waltham, MA, USA) in 0.1 M sodium phosphate buffer (pH 8.0) containing 10 mM SDS for 4 h. Solubilized proteins were incubated with cobalt resin (Thermo Fisher Scientific) or Ni-NTA resin (Qiagen, Crawley, UK) for 4 h at room temperature for shaking. His-tagged protein was passed through filter column (Bio-rad, Richmond, CA, USA) and eluted from the 0.1 M sodium phosphate buffer (pH 6.0) with 10 mM SDS by gravity flow. Eluted protein was performed to western blotting and Coomassie blue staining.
E coli bl21 strains
Manufactured by RBC Bioscience
The E. coli BL21 strains are a commonly used laboratory strain of Escherichia coli bacteria. They are designed for the expression of recombinant proteins. The BL21 strains are widely utilized in various research and biotechnology applications due to their ability to efficiently produce target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Urea, by Merck Group (6 mentions)
Isopropyl β d thiogalactoside iptg, by Duchefa Biochemie (3 mentions)
Ni nta his binding resin, by Merck Group (3 mentions)
Cobalt resin, by Thermo Fisher Scientific (1 mentions)
Mwco slide a lyzer dialysis cassette, by Thermo Fisher Scientific (1 mentions)
Ni nta resin, by Qiagen (1 mentions)
4m urea, by Merck Group (1 mentions)
Ni nta his bind resin, by Merck Group (1 mentions)
Isopropyl β d thiogalactoside, by Duchefa Biochemie (1 mentions)
5 protocols using e coli bl21 strains
1
Recombinant Protein Purification from E. coli
2
Recombinant MIF Protein Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant MIF protein was purified from Escherichia coli, as previously described [23 (link),24 (link)]. Briefly, BL21 E. coli strains (RBC Bioscience, Taipei City, Taiwan) were transformed with pET28a-MIF in order to express and purify the fusion protein. Bacterial cultures were induced with 0.4 mM isopropyl β-d -thiogalactoside (IPTG, Duchefa Biochemie, Haarlem, Netherlands). Next, bacterial cultures were sonicated for 10 min. at 4 °C, and then centrifuged at 1600× g for 20 min. at 4 °C. Pellets containing His-tagged MIF (His-MIF) were resuspended in binding buffer (0.5 M NaCl, 20 mM Tris-HCl, 5 mM imidazole) containing 4 M urea (Sigma Aldrich, St. Louis, MO, USA). His-MIF was purified while using Ni-NTA His•Bind Resin (Merck, Darmstadt, Germany) and subsequently eluted using elution buffer (300 mM NaCl, 50 mM sodium phosphate buffer, and 250 mM imidazole) containing 4 M urea. Finally, MIF protein was dialyzed in order to remove imidazole, residual salts, and urea.
3
Recombinant p24 Protein Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant p24 proteins were purified from E. coli as previously described (59 (link)) with minor modification. For the expression and purification of the fusion protein, E. coli BL21 strains (RBC Bioscience, Taipei City, Taiwan) were transformed with pET23a-p24. Protein expression was induced by adding 0.4 mM isopropyl β-d -thiogalactoside (Duchefa Biochemie, Haarlem, Netherlands). Bacterial cells were harvested and disrupted by sonication on ice for 10 min. Sonicated lysates were centrifuged at 1,600 × g for 20 min at 4°C, and the pellets containing p24 protein were resuspended in binding buffer containing 4 M urea (Sigma Aldrich, St. Louis, MO, USA). The proteins were purified using Ni-NTA His binding resin (Merck, Darmstadt, Germany) and eluted with elution buffer (300 mM NaCl, 50 mM sodium phosphate buffer, 250 mM imidazole) containing 4 M urea. Purified proteins were dialyzed serially against the elution buffer to remove imidazole, urea, and residual salts. Purity of p24 protein was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 12% gel). The gel was visualized using Coomassie brilliant blue staining methods (60 (link)) (Figure S9 in Supplementary Material).
4
Recombinant Tuberculosis Antigen Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant Ag85B, CFP10, ESAT-6, and TBCM proteins were purified from Escherichia coli as previously described [18 (link),19 (link)]. Briefly, E. coli BL21 strains (RBC Bioscience, Taipei City, Taiwan) were transformed with pET28a-Ag85B, pET28a-CFP10, pET28a-ESAT-6, or pET28a-TBCM for the expression and purification of each fusion protein. Protein expression was induced by adding 0.4 mM isopropyl β-D -thiogalactoside (IPTG, Duchefa Biochemie, Haarlem, The Netherlands). Cultured bacterial cells were disrupted by sonication (10 min, 4 °C), and the resultant lysates were centrifuged (1600× g, 20 min, 4 °C). The pellets containing each protein (Ag85B, CFP-10, ESAT-6, or TBCM) were resuspended in binding buffer containing 4 M urea (Sigma Aldrich, St. Louis, MO, USA). Each protein was purified with Ni-NTA His binding resin (Merck, Darmstadt, Germany) and eluted with elution buffer (300 mM NaCl, 50 mM sodium phosphate buffer, and 250 mM imidazole) containing 4 M urea. Purified proteins were dialyzed serially against the elution buffer to remove imidazole, urea, and residual salts.
5
Recombinant Ag85B and p24 Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant Ag85B and p24 proteins were purified from E. coli as previously described45 (link) with minor modification. For the expression and purification of fusion protein, E. coli BL21 strains (RBC Bioscience, Taipei City, Taiwan) were transformed with pET23a-Ag85B or -p24. Protein expression was induced by adding 0.4 mM isopropyl β-D-thiogalactoside (IPTG, Duchefa Biochemie, Haarlem, Netherlands). Bacterial cells were harvested and disrupted by sonication on ice for 10 min. Sonicated lysates were centrifuged at 1600 ×g for 20 min at 4 °C, and the pellets containing Ag85B and p24 proteins were resuspended in binding buffer containing 4 M urea (Sigma Aldrich, St. Louis, MO, USA). The proteins were purified using Ni-NTA His binding resin (Merck, Darmstadt, Germany), and eluted with elution buffer (300 mM NaCl, 50 mM sodium phosphate buffer, 250 mM imidazole) containing 4 M urea. Purified proteins were dialyzed serially against the elution buffer to remove imidazole, urea and residual salts.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!