User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2009/07/31/Metal chelate chromatography

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Overview

So apparently it is straight forward to make recombinant proteins in e. coli. Getting the proteins you made the e. coli produce for you purified, has lots of steps associated to it and one of them is metal chelation chromatography. Typically people put his-tags on the proteins of interest. These his-tags have a "high" affinity for nickel ions and thus can be purified from your sauce if put through a column containing a nickel resin.

Questions

  • Adding the his-tag to the kinesin means we will have a his-tag on the kinesin after purification. Do we want to remove this tag?
  • If we do want to remove this tag, then do we want to remove it while it is attached to the column or after it has been eluted? Is there a preference? Does one or the other work better?
  • Why do elution buffers have more imidazole in them than wash buffers?
    • Answer: This is a wording issue for me. The wash buffer is used to wash the column and not to wash the proteins from the column. Elution is the word used to remove the proteins from the column and thus this is why the elution buffer has more imidazole.
  • His-tags have an imidazole structure in them. This means that free imidazole and his-tagged proteins will compete with binding to the nickel resin. If it does, then why is there so much imidazole in the elution buffer.
    • Answer: As above, the answer is in the naming convention.
  • Molecular Cloning (MC) says that it is useful to add 1 or 2 glycine residues in between the his-tag and the protein of interest. Why would you want to do this? Is it useful for purification of kinesin?
  • What is the off rate for imidazole and nickel?
  • MC says that any chelator will interfere with the his-tag affinity to the nickel resin. This means that EDTA and EGTA cannot be in solution when purifying the protein. Why do we not use EDTA for elution then? What is the affinity for imidazole and nickel?
  • MC says that EDTA will strip off nickel from the agarose resin. What is the affinity for EDTA and nickel?
  • MC says that BME is useful for metal chelation chromatography. What's so special about BME? Why would we use it at all?
  • Koch question: Our protocol has a considerable difference in the concentration of NaCl2 used in the wash and elution buffer. Why is this the case?
  • All the buffers in our protocol have ATP in them. Is the ATP used to keep kinesin active? I think I've asked myself this question before but I cannot remember it but, does kinesin inactivate irreversibly if it doesn't have ATP around?
  • Why do we use NaPi? Is it for pH-ing?
  • MC says to use 3 buffers in the nickel column. One is a "binding buffer" used to remove all imidazole from the column. Two is a "wash buffer" that is used to get the protein of interest into the column. And, three is the "elution buffer" which is used to get the protein out of the column. Our protocol doesn't have a "binding buffer". Koch question: Is there a specific reason for this?
    • Answer: Having some imidazole in the wash buffer will help prevent any non specific protein binding. This is why in our protocol we have a small amount of imidazole in the wash buffer.
  • MC states that the "binding buffer" and "elution buffer" should be at different pHs. Why is this the case?

Some basics

  • His-tags are usually placed on the N or C terminus for the protein of interest.
  • Few natural proteins will bind to nickel ions. This is why this chromatography technique is useful. However, some proteins will non selectively bind to the nickel ions. This is why you put a small amount of imidazole in the column when you use the wash buffer.
  • Do not use EDTA or EGTA in any buffer when you are fractionating the protein of interest. You do this so that the chelators don't strip off any of the nickel ions from the resin.
  • Sigma suggests that the column is stored with 20% ethanol to prevent bugs from growing in it.
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