& Data Analysis
Protein gel analysis
Keeping a lab notebook
Writing research papers
Dimensions & units
Using figures (graphs)
Examples of graphs
Principles of microscopy
Solutions & dilutions
Fractionation & centrifugation
Radioisotopes and detection
Working with Stock Solutions
We define a stock solution as a concentrate, that is, a solution to be diluted to some lower concentration for actual use. We may use just the stock solution or use it as a component in a more complex solution. We refer to the solution that we end up using as a working solution. If you are comfortable making dilutions then you can appreciate the many advantages of working with stock solutions. Although it is never absolutely necessary to use a stock solution, it is often impractical not to use them. Stock solutions can save a lot of time, conserve materials, reduce needed storage space, and improve the accuracy with which we prepare solutions and reagents. Here are several illustrated types of applications using stock solutions.
Storing a solution as a concentrate
We frequently run protein gels using the Laemmli method of SDS-PAGE. The driving force that separates proteins in a gel is an electric field. To produce the field both ends of a gel and respective positive and negative electrodes are immersed in a solution that we call electrode buffer, and a typical class goes through 30 liters or so of the stuff. It would be nice to make up the buffer in advance, but 30 liters is a lot to store, and it has to be sterilized to be stored more than a few days. Sterilizing 30 liters of buffer takes a long time.
We typically refer to the strength of a stock solution by a number followed by the times symbol x. For example, a stock solution that is concentrated by a factor of 10 is called a 10 times concentrated stock, a 10x concentrate, a solution of 10x strength, or simply a 10x solution. A normal working solution is a 1x, or normal strength solution.
A Stock solution as a component of a complex working solution
A stock solution can be mixed along with other components, including other stock solutions, to make a working solution. For example, to study respiration by isolated mitochondria we need to suspend them in a complex medium consisting of 70 mM sucrose, 220 mM mannitol, 2 mM HEPES buffer, 5 mM magnesium chloride, 5 mM potassium phosphate, 1 mM EDTA, and 0.1% fatty acid free bovine serum albumin, pH 7.4. It is practical to weigh out and dissolve the sucrose, mannitol, HEPES, and albumin, however we run into complications with the magnesium chloride, EDTA, and potassium phosphate.
Magnesium chloride is extremely hydroscopic, that is, the dry chemical accumulates moisture from the atmosphere when stored on a shelf. Sometimes one will open even a new bottle, and depending on where it has been and for how long you might find your crystals immersed in a semi-liquid slurry. Because we don't know how much water has accumulated and because water adds weight, it is impossible to obtain an accurate concentration of magnesium chloride by weighing out the chemical unless it has been stored in a very effective dessicator. A trick is to purchase a known quantity of the material, for example 500 gms, and without even weighing it to prepare the entire lot as a stock solution. You can prepare, for example, a liter of 0.5M (500 mM) magnesium chloride. To use the stock to prepare respiration medium, simply include 10 ml of stock solution per liter of working solution. For this application, then, 0.5M magnesium chloride is a 100x stock.
Ethylene diamine tetraacetic acid (EDTA) as a free acid will not go into solution without bringing the pH to near neutrality. Unfortunately, because the material continues to reduce the pH as it dissolves, dissolving a quantity can be a tedious process. EDTA salts tend to dissolve more readily, but you would have to keep two different salts for sodium based and potassium based solutions respectively. In addition, we might use EDTA in a concentration as low as 0.1 mM in a small volume. It is usually easier to deliver a small volume of liquid accurately than to accurately weigh out and deliver a small amount of dry chemical. A concentrated stock of EDTA can be prepared using either NaOH or KOH to adjust pH, to be available whenever a solution requires EDTA as a component. For example, our respiration medium is potassium based so we might prepare 100 mM EDTA and use 1 ml of the stock per liter of working solution.
The issue with phosphate buffers calls for a write-up of its own.
Working with phosphate buffer stocks
When we include a phosphate buffer in a solution we choose a salt that is compatible with the intended use of the solution. For example, the principal positive electrolyte inside cells is potassium, while the principal extracellular cation is sodium. Because mitochondria are intracellular organelles we suspend them in solutions buffered with potassium salts. The name potassium phosphate refers to a family of inorganic salts, including dibasic and monobasic potassium phosphate. The formula for the dibasic form is K2HOP4 and for monobasic it is KH2PO4. The monobasic form is actually quite acidic. We can make a stock solution of potassium phosphate at a desired pH by mixing monobasic and dibasic stocks. A practical concentration for each stock is 100 mM.
For a pH near neutrality we start by stirring dibasic solution and slowly adding monobasic stock while monitoring pH, stopping when we reach the desired value. The concentration of phosphate ion remains 100 mM because that was its concentration in both of the "parent" stock solutions. To use the buffer as a component of respiration medium we simply add an appropriate volume of 100 mM stock solution before bringing the medium to final volume. What volume will you need to add to obtain a final concentration of 5 mM phosphate? What is the strength of the concentrate?
Biologists often work with Ringers solutions, relatively simple buffers that contain a limited mixture of inorganic and perhaps some organic salts. A marine biologist or developmental biologist might work with artificial seawater (ASW), also a relatively simple solution of common salts. The composition of Ringers or ASW may be varied for experimental purposes. For example, the concentrations of potassium and sodium ions both have profound individual effects on the level of the membrane potential in muscle cells immersed in Ringers. It would be inconvenient to weigh out different amounts of dry potassium and/or sodium over and over as different solutions are required. On the other hand, it is a quick and simple matter to pipet different volumes of stock solutions before bringing a Ringers solution to final volume.
A formula for frog Ringers is 0.65% NaCl, 0.014% KCl, 0.012% CaCl2, 0.1% NaHCO3. Notice that to prepare a liter of working solution would require weighing as little as 0.1 gm of dry chemical, creating a high potential for mistakes. On the other hand you could prepare a liter or more of 10x NaCl stock by weighing, dissolving, and bringing to 1 L volume 65 gms NaCl. You could prepare a liter each of 100x KCl, CaCl2, and NaHCO3 by weighing, dissolving, and bringing to volume 14, 12, and 10 gms per liter of each respective chemical. Not only is it more accurate to weigh a larger amount of material, but now you can change the Ringers formula at will. For example, for normal Ringers you would include 10 ml KCl stock per liter working solution. Suppose you want to raise the concentration of potassium five-fold. Then include 50 ml KCl stock per liter.
Working with samples
Suppose that you want to test the effectiveness of a substance in preserving enzyme activity. You plan to mix enzyme with preservative in aliquots and store them frozen, conducting an assay periodically to see how long the enzymes remain functional. To give your new preservative a good test you conducted a dozen enzyme preparations and have each prep in solution. You have measured the protein concentration of each enzyme solution.
Suppose also that you have stored your preservative as a 5x concentrate. To facilitate the assays and keep the storage conditions uniform you want to prepare each enzyme solution to the same protein concentration (say, 1 mg/ml) in 1x preservative. How do you go about deciding what volumes to mix together?
First, decide what approach to take to diluting the enzyme preparation itself. Do you want to prepare the whole batch to a final concentration of 1 mg/ml or do you want to prepare a specific volume to 1 mg/ml and save the rest? Recall how to use the C1V1 = C2V2 relationship to set up each type of problem. Next, apply the relationship to determine your unknown quantity, which is either the final volume of your solution (V2) or the volume of enzyme solution to start with (V1). Separately, decide how much 5x preservative solution to include in your diluted enzyme solution to end up with 1x preservative.
For example, let the protein concentration of one enzyme batch be 12 mg/ml. Suppose that you have 3 ml and want to bring it all to 1 mg/ml. Using the "C,V" equation you can calculate the desired final volume to be 36 ml. After all, 3 ml of 12 mg/ml contains 36 mg protein, and 36 mg protein in 36 ml volume give you 1 mg/ml. You can arrive at the same conclusion by treating the enzyme solution as a stock solution. A concentration of 12 mg/ml is a 12x stock when your working concentration is 1 mg/ml.
Before bringing your enzyme solution to volume, factor in the preservative stock. You will prepare a final volume of 36 ml of enzyme-preservative. The preservative concentration is 5x. Then 1/5 of the final volume must be preservative stock, and 1/5 of 36 ml is 7.2 ml.
So, the answer to your problem is to combine 7.2 ml preservative solution with 3 ml enzyme solution and bring the final volume to 36 ml with water or buffer.
What do the ratios mean?
Suppose someone asks you to "prepare a one to ten dilution of solution X." Does it mean take one part solution X and add ten parts water, or does it mean take one part solution X and bring the volume to a total of ten parts? A biologist would likely apply the second definition to a buffer or reagent solution. In another discipline, though, the former definition might be more relevant. Even in biology, we often prepare complex media as weight-to-volume (w:v) instead of weight-in-volume (w/v), that is, we add a prescribed mass of material to a prescribed volume of water instead of mixing the materials and bringing the mixture to a prescribed final volume.
To avoid confusion you might say "please prepare a one to ten dilution of solution X, weight-in-volume," or if you want to bring materials together in a precise proportion, say "please prepare a one to ten dilution of solution X, weight-to-volume." In the latter case it might be less confusing just to say "please combine one part solution X with ten parts water."