How to prepare Tris-HCl buffer for protein crystallization? What does 33mM mean? - (Mar/20/2012 )

sorry I fail to understand the following recipe to crystallize a protein:

450 mg protein was dissolved in 10ml of 33 mM Tris-HCl buffer (pH 5.8) in the presence of 4 mM CaCl₂ and 90 mM benzamidine.

2.1 M(NH₄)₂SO₄ was dissolved in the same buffer. Equal volumes of protein and precipitant solution were mixed to obtain a solution with 22.5 mg/ml trypsin and 1.05 M Ammonium sulfate. ...



I really don't know which amounts of the different agents I have to use. Does it mean that I prepare the Tris-HCL Puffer by using 0.033 mole Tris/Tris-HCL per litre and the use 10 ml of that? Do I have to solute CaCl₂ and benzamidine in 10ml Tris-HCl buffer to concentrations of 0.004 mole/L and 0.09 mole/L? Do I have to adjust the pH to 5.8 before adding CaCl₂ and benzamidine or after that?

And do I have to solute 2.1 mole ammonium sulfate per litre (so 0.01 * 2.1 mole for 10ml) to prepare the precipitant solution? What about the volume expansion due to ammonium sulfate? I mean that changes the concentration of the other agents right? so do I have to weigh in them to the concentration mentioned in the recipe after or before adding ammonium sulfate?


I don't understand the recipe at all: what might be the reason to use Tris at the pH value of 5,8 where it it not a buffer at all?

It is the first time I have to work on that, so hopefully someone can tell me what to do?

You need to prepare a 0.033 Mol/L solution of tris, pH it to 5.8, adding CaCl2 and benzamidine to the solution as well. You may be able to add these as separate components later, using more concentrated solutions to dilute to the correct concentration (e.g. prepare a 100 mM tris solution, and a 10 mM CaCl2 solution, etc... then mix these components in the appropriate amounts with some water to give the desired concentrations). I would prepare larger volumes than you need, not just because it makes it easier to work with them, but also because you are likely to have to try this experiment several times.

You are also correct about adding the ammonium sulphate, though this one you might want to add as a powder/crystalline compound to the prepared buffer. I wouldn't worry about the volume expansion, I don't think it will be too significant. if you are worried, you can prepare *any* solution separately, it just takes more time/bottles.

I have no idea why they used tris at pH 5.8, they would have been better off using MES or MOPS, though they might interfere with the protein.

Thank you for your reply. Sorry I don't get it completely.

I thought that I have to add CaCl2 and benzamidine also as crystalline compounds in 10 ml of tris-buffer, so 5.8 mg CaCl2 and 108.1 mg benzamidine. Then the total volume is 10 ml and contains 33mM tris, 4mM CaCl2, and 90 mM benzamidine. Is that right or do I have to add CaCl2 and benzamidine in addition to the 10 ml Tris, so that the final volume is > 10 ml? Would you adjust the pH before or after adding CaCl2 and benzamidine? Because I think benzamidine is basic so the pH will not be 5,8 after I added that to Tris-Hcl 5,8.

And the same issue with the precipitant solution: do I need to adjust the pH after adding Ammonium sulfate or before?

Thanks a lot!

What I would do is make separate, more concentrated solutions of each of the reagents and mix them, along with an appropriate amount of water to make 10 ml. This way, you will have a tris solution of pH 5.8, a benzamidine solution and a CaCl2 solution ready for use each time, all you need to do is dilute. The only problem would be if one of the components degrades upon storage, as benzamidine may do - you should check this.

However, there is no reason why you couldn't add the compounds in a crystalline form to your tris solution, unless there are solubility issues.

Follow the concentrations you have been given, these may be important for the protocol, so don't make the volume more than the 10 ml if you are adding the crystals, as this will alter the concentration of the other reagents.

I would pH before adding the other compounds, this is why the reagents are written Tris (pH 5.8), CaCl2..., to indicate that it is the tris that is pH adjusted not the final solution., CaCl2 won't affect the pH, but depending on the form of benzamidine you have, it may alter the pH. This is probably irrelevant to your experiments though, so I wouldn't worry about it.

thank you for your detailed reply.

Benzamidine is not stable at ambient temperature so I have to freeze the solution and store at -20 °C.

Do you think that I have to mix 10ml of protein-solution and 10ml precipitant-solution, or is that unusual for batch crystallization technique?

No idea, never crystalized proteins before.

the formulation you describe in your original post says that you mix equal volumes of protein solution and precipitation solution.

the volume of protein solution should be determined by the concentration of the protein solution and how much protein you want to crystallize.