• Question: When you create proteins using E.coli you have to separate the desired protein from the other proteins made by the E.coli. How do you separate these proteins and know you have obtained the desired one? Would it involve using a centrifuge and fluorescent markers that attach to areas of the desired protein that are unique?

    Asked by seanj to Jo on 25 Jun 2014.
    • Photo: Jo Nettleship

      Jo Nettleship answered on 25 Jun 2014:

      Hi seanj,
      Usually we tag the protein we are interested in with 6 histidine residues in a row. You can then use a special resin with Nickel attached to it to purify your protein away from the E. coli proteins. Basically, the His6 tag binds to the nickel which is attached to the beads of the resin. You can then wash the resin to remove the E. coli proteins. At the end you elute your protein with a chemical called imidazole which out-competes your protein for the nickel. You then get your protein nice and pure.
      For fluorescent markers, we often use GFP (green fluorescent protein) marker to label proteins to see where they are in the cell. It is quite big though so we prefer to use the His6 tag when purifying proteins so that the protein is more “native” or like it is in the human body.