User:Andy Maloney/Kinesin & Microtubule Page/PEM buffer

Chemicals

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 pK_a 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. 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

Be careful with this. It is a strong base. NaOH is again hygroscopic and thus likes to pull moisture from the atmosphere. I store it in its original container and in the desiccator. When ever I open the jar and close it, I 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. I like to make up 1 Normal solutions of this and use it in this form.

When making a 1 N solution, I typically store it in a glass jar. I sometimes will also make up a 12 N solution and dilute it from that but, this is not necessary.

Recipe

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

Notes

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 your 1 N NaOH stock solution. I had previously been doing this, however, I think I like weighing out an amount of NaOH that will be less than 125 mM concentration in my final solution and just adding the appropriate amount of the 1 N solution to get it to the ~120 mM.

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.

If, you make the solution too basic, just add small amounts of HCl to get the pH right.

References

There is quite a debate about who came up with the PEM buffer and what the initials BRB80 mean. BRB80 is another name for PEM. Below are different recipes from other labs.

• Hancock
  • 80 mM PIPES
  • 1 mM MgCl₂
  • 1 mM EGTA
  • pH 6.85
• Cold Spring Harbor Labs
  • 100 mM PIPES
  • 2 mM EGTA
  • 1 mM MgSO₄
  • pH 6.95
• Sigma Aldrich
  • 100 mM PIPES
  • 5 mM EGTA
  • 2 mM MgCl₂ · 6H₂O
  • pH 6.8
• Bayer College of Medicine
  • 80 mM K-PIPES
  • 5 mM EGTA
  • 2 mM MgCl₂
  • pH 6.8
• Yu-li Wang from Carnegie Mellon This is a pdf file.
  • 100 mM PIPES
  • 1 mM EGTA
  • 0.5 mM MgCl2
  • pH 6.9
• Tedeschi of Italy
  • 85 mM Pipes
  • 10 mM EGTA
  • 1 mM MgCl₂
  • pH 6.94
• Steve Gross of U. Minnesota with Steve Block
  • 80 mM Pipes
  • 1 mM EGTA
  • 4 mM MgCl₂

As you can see, there is no consensus on what BRB80/PEM is. It seems that the term is used for just about any buffer that has PIPES, EGTA, and some form of Magnesium in it.