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Chazin Lab Protocol for Limited Proteolysis

notes:

Written 10-03-02 by Melissa Stauffer

Purpose

This protocol describes the general procedure for setting up a limited proteolysis reaction. An experiment like this is useful for determining the sizes, timed order of appearance, and relative amounts of fragments produced from the protein of interest by digestion with a protease. From the results, you may be able to infer the presence of stable subdomains. It is also possible to obtain evidence of binding of one protein to another by running two identical sets of proteolysis experiments, one with the putative target, and one without. If the rate of digestion of the protein is slowed by the addition of the putative target, one can conclude that they interact with each other.

The basic procedure involves exchanging the protein of interest into an appropriate buffer system and diluting to a desired volume. A protease is then added to start the reaction, and aliquots of the reaction are removed at specific time points. The aliquots are then boiled to inactivate the protease and the results are analyzed by SDS-PAGE.

Variables to consider

  1. Amount of protein
  2. Amount of protease
  3. Type of protease
  4. Reaction volume
  5. Time increments
  6. Gel sample size
  7. Staining method
Comments and suggestions corresponding to each of these variables are found below:

Considerations

  1. Determine the amount of protein you want to expend on the experiment. This will automatically determine the type of gel staining you use. In my experience, the following chart is a good estimate:

    2. ug protein » ng protease » breakdown in hours » silver stain;
    ug-mg protein » ug protease » breakdown in minutes » coomassie stain.
  2. The amount of protease should be about 1000x less than the amount of protein in mass units. This quantity can be adjusted so the rate of proteolysis fits the desired time frame. Protease solutions can be made in water, but they may lose activity over time, so make the stock solution from solid powder as close to the start time of the experiment as possible.
  3. Choose several different types of protease for the first try. We routinely use chymotrypsin, trypsin, and proteinase K to probe a wide range of specificity.
  4. The reaction volume should be designed to provide the appropriate number of gel sample aliquots. For example, 80 mL will give eight 10-mL gel samples. Be sure that each aliquot will contain an observable amount of protein for the staining method you choose.
  5. Time increments can be chosen based on what is convenient. As stated above, the amount of protease can be adjusted so the reaction goes faster or slower. The total reaction time can be as short as a single hour or as long as a whole day (or more), depending on how much detail you need to see. We usually remove aliquots at 5, 10, 30, and 60 minutes, then each hour or two after the first hour until the reaction has ended.
  6. Gel sample size can, of course, be adjusted to contain an observable amount of protein. This is an easy way to make up for miscalculations in protein concentration, since you will often be thinking in total quantities of protein and protease.
  7. The staining method is up to the user, but I prefer using regular Coomassie staining if I have enough protein. Silver stain is so sensitive that it is difficult to separate impurities from degradation products.

Example

The following gels were obtained from digestion of ~400 ug protein with ~20 ng proteinase K in 20 mM Tris, pH 8.0, 1 mM BME. Total reaction volume was 80 uL, and 10 uL gel samples were remoived. The gels were stained with Coomassie.
As you can see, the middle gel shows that the addition of Rad51N protein reduces the rate of degradation of RPA70AB, thus implying a physical interaction between them.