# I have 100 mg of a proteinase K lyophilized powder and I need to make it to a working concentration of 25 mg/mL

So I know that I need to add $4~\mathrm{mL}$ of solution to this powder, which will give me a concentration of the enzyme at $25\ \mathrm{mg/mL}$. What I do not understand is how to decide how much of each of these "ingredients" (below) to add. I have $\ce{TrisHCl}$ and $\ce{CaCl2}$ in granular form ($M = 157\ \mathrm{g/mol}$ and $111\ \mathrm{g/mol}$, respectively) and 100 % glycerol solution. Will I have to make a large batch of Tris and $\ce{CaCl2}$ just so I can actually weigh out the needed amount?

The specifications for the working solution are:

$10\ \mathrm{mM}\ \ce{Tris}$
$1\ \mathrm{mM}\ \ce{CaCl2}$
30 % glycerol
Final $\mathrm{pH} \approx 8$ (use $\ce{HCl}$ if necessary)
MilliQ $\ce{H2O}$ to volume

Simply adding 4 mL of solution to 100 mg of enzyme is not an accurate way of preparing this solution. I am going to describe what to do from an analytical chemist's point of view. If you don't need this level of precision, you could do this following the same steps without volumetric glassware.

The first thing you need to do is decide what volume of your 25 mg/mL solution you need. Ideally, you will have a volumetric flask corresponding to this volume. For now, let's go with the 4 mL that you mentioned.

Next you will need to make a stock solution of your buffer. This will include all of the components except for the enzyme:

1. Decide what volume of buffer to make. You will need to make at least enough for your working solution, and again you will want to use a volume for which you have a volumetric flask. Also, you want to be weighing out reasonable amounts of your reagents (because weighing out <10 mg of something can really suck). Let's assume you're making 1 L. You're reagents are cheap anyway and the larger scale will make everything easier.

2. Calculate how much $\ce{TrisHCl}$ and $\ce{CaCl2}$ you will need to obtain the desired concentrations in 1 L of solvent.

3. Weight out the $\ce{TrisHCl}$ and $\ce{CaCl2}$ and add it to your volumetric flask.

5. Fill nearly to volume with water. You need everything to be in solution so you can adjust the pH, but you also need to leave some room to work with to do the actual adjustment. I'd shoot for approximately 80% full.

6. Adjust the pH by adding concentrated $\ce{HCl}$ dropwise.

7. Fill to volume with water and thoroughly mix the solution.

Now that you have your buffer solution, you just need to weigh out the appropriate amount of your enzyme and dissolve it in your buffer. From the example above, you would weigh out 100 mg of the enzyme (don't just assume you have 100 mg, even if the bottle says so) and dissolve it in your buffer using a 4 mL vol. flask.*

* If you don't have a 4 mL vol. flask and you actually need all 100 mg of your enzyme in this solution, you will need to mess around with dilutions. Start by by creating a 50 mg/mL (2 mL vol. flask) or 100 mg/mL (1 mL vol. flask) solution.

• To get the correct concentration of $CaCl_2$, you would weigh just under 1 mg. That's difficult to do. I would weigh 50 mg of $CaCl_2$ and dissolve into 50 mL, and use 1 mL of that solution. Also, pipettes are very useful for small volumes.
– LDC3
Commented Jan 16, 2015 at 4:32
• @LDC3 Yeah, the buffer prep should really be scaled up. I didn't actually do the math for the concentrations, but it's pretty obvious once you think about it.
– Kyle
Commented Jan 16, 2015 at 4:35