Top 10 one shot

We initially carried out a protein expression pilot as a preliminary study to identify HSP70-expressing colonies by inoculating a single colony into test tubes containing Luria-Bertani (LB) broth culture medium (3 mL). Once the colonies had been identified, they were cultured in larger volumes of LB medium.
The protein was expressed in E. coli strain TOP10 One shot ® (Invitrogen). Briefly, cells were grown in LB broth culture with 100 µg/mL ampicillin at 37°C to an optical density at wavelength 600 nm (OD 600 ) of 0.5-0.7, measured on an iMark™ Absorbance Microplate Reader spectrophotometer (Bio-Rad, Hercules, CA, USA). rHSP70 protein expression was induced in LB medium containing 1 mM isopropyl-thio-D-galactoside for 4 h at 37°C, agitated at 200 rpm. The HSP70 protein was purified by Ni-NTA nickel resin affinity chromatography (Qiagen Biotechnology Brazil Ltd.) under denaturing conditions, according to the manual instructions, whereby guanidine was used to lyse and bind the proteins in the resin and 8 M urea was used for washing and elution. The final purified solution was dialyzed in PBS and subjected to 12% polyacrylamide gel electrophoresis. The protein concentration was determined using a Pierce ® BCA Protein Assay Kit (Thermo Scientific, San Jose, CA, USA).