Prokaryotic expression and protein purification

An in-frame insertion of the cDNA of the full human Smad1 gene with a glutathione S-transferase (GST) tag into the plasmid pGEX-4 T-1 was performed, and the resulting recombinant plasmid was transformed into Escherichia coli strain BL21. GST-tagged Smad1 protein expression was induced using 1 mM isopropyl-β-d-thiogalactopyranoside, and the resulting protein was purified with glutathione Sepharose 4B beads (GE Healthcare) according to the manufacturer’s instructions. Protein was digested by thrombin and dialyzed against buffer I [10 mM tris-HCl (pH 7.5), 1 mM dithiothreitol (DTT), and 0.2 M NaCl] at 4°C overnight. Proteins were brought to a final concentration of 200 μg/ml.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.