Koch Lab:Protocols/Kinesin/GTP suspension

There are many opinions on what you should do with GTP storage. Since all of our buffers contain PEM, our GTP will also be stored in it. One caveat is that PEM has EGTA in it. EGTA chelates metal ions and will thus chelate the magnesium in our buffer from solution. For proper tubulin polymerization to occur, there must be magnesium in solution. Thus, Koch has asked that GTP be stored in PEM with the addition of 1 mM MgCl2.

|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC GTP comes packaged nicely in 10 mg jars so we do not have to weigh it out. It is highly toxic so use caution when opening the jar. You may want to do this in the hood to prevent any inadvertent inhalation of GTP dust. In order to get all the GTP from the bottle into solution, you are going to have to slosh the contents around after you have added the PEM and MgCl2. If you are very careful, you can take the tube out that has the GTP in it (see below picture).



You can then wrap some Parafilm on the top and carefully get the GTP that may be stuck on the sides to go into solution. After you have done this, transfer it into a microcentrifuge tube and flash freeze it. It should be stored in the -20˚C freezer.