Koch Lab:Protocols/Kinesin/PEM

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PEM is our base buffer and we use it for just about everything in our microtubule experiments. Below are some comments about the chemicals used and the recipe for making PEM.

Contents

PIPES

Somethings to note about PIPES is that it is an acid. Don't get this stuff on your skin because it will irritate it. We use acid PIPES because there was a discussion with Koch about what type of ions are needed in solution for motility to work. Some say that you need potassium and others, sodium. Since we have a free acid form of PIPES, we can add either NaOH or KOH to pH the buffer to PIPES's pKa of 6.89 thus adding the appropriate counter ion we want.

PIPES should be stored in the desiccator at room temperature in its original container.

EGTA

EGTA is also an acid so be careful. The reason we use the acid form is for the same reason we use the acid form of PIPES. It should be stored in its original container in the desiccator.

MgCl2

MgCl2 is very hygroscopic. This means it really likes to be in water and it will pull moisture from the atmosphere so it can be in it. For this reason, we use a 1 M solution of MgCl2 in water purchased directly from Sigma. This way we do not have to fuss around with trying to weigh out an appropriate amount of MgCl2, since what we weigh would also include water.

MgCl2 should be stored in its original container at room temperature. You do not have to desiccate it since the MgCl2 is in solution already.

NaOH

NaOH is again hygroscopic and thus likes to pull moisture from the atmosphere. Store it in its original container and in the desiccator. After opening the jar, blow in nitrogen so it is stored in a nitrogen rich environment (i.e. no moisture). It is a strong base so be careful when handling it.

Make up 1 Normal solution and store it in a glass jar.

Recipe

  • 80 mM PIPES
  • 1 mM EGTA
  • 1 mM MgCl2
  • ~ 125 mM NaOH
  • pH 6.89

Always put PIPES and EGTA into water and not the other way around. Once you have all of your PIPES and EGTA in some water, go ahead and add around 120 mM of NaOH. You will see that before the NaOH is added, PIPES and EGTA will not go into solution. After adding it, it will. Keep adding small amounts of NaOH into solution until all the PIPES and EGTA is dissolved (the total amount is approximately 125 mM). If you are lucky, you will get really close to the pH that the buffer needs to be at once all the PIPES and EGTA is in solution. Add the remaining amount of water needed and pH the solution till you get it to a pH of 6.89.

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