Chazin Lab Protocol for Limited Proteolysis
Written 10-03-02 by Melissa Stauffer
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
by running two identical sets of proteolysis experiments, one with the
putative target, and one without. If the rate of digestion of the
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
is then added to start the reaction, and aliquots of the reaction are
at specific time points. The aliquots are then boiled to inactivate the
protease and the results are analyzed by SDS-PAGE.
Variables to consider
Comments and suggestions corresponding to each of these variables are
Amount of protein
Amount of protease
Type of protease
Gel sample size
Determine the amount of protein you want to expend on the experiment.
will automatically determine the type of gel staining you use. In my
the following chart is a good estimate:
ug protein » ng protease » breakdown in hours »
ug-mg protein » ug protease » breakdown in minutes
The amount of protease should be about 1000x less than the amount of
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,
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.
Choose several different types of protease for the first try. We
use chymotrypsin, trypsin, and proteinase K to probe a wide range of
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
Be sure that each aliquot will contain an observable amount of protein
for the staining method you choose.
Time increments can be chosen based on what is convenient. As stated
the amount of protease can be adjusted so the reaction goes faster or
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.
Gel sample size can, of course, be adjusted to contain an observable
of protein. This is an easy way to make up for miscalculations in
concentration, since you will often be thinking in total quantities of
protein and protease.
The staining method is up to the user, but I prefer using regular
staining if I have enough protein. Silver stain is so sensitive that it
is difficult to separate impurities from degradation products.
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
was 80 uL, and 10 uL gel samples were remoived. The gels were stained
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