User:Andy Maloney/Kinesin & Microtubule Page/PEM-G

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Chemicals

PEM

See the above page for more information.

GTP

Originally we purchased GTP from Cytoskeleton. Unfortunately, Cytoskeleton doesn't tell us what exactly it is we purchased and they have mixed protocols about how to suspend GTP into solution.

Since we have a salt form of GTP, it goes into solution very easily. I have it stored in a secondary container that is filled with desiccant and under a nitrogen environment in the -20˚C freezer. I also wrapped Parafilm around the cap to ensure that no moisture gets in.

MgCl2

Recipe

Notes

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.

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).

GTPsuspension.jpg

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.