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Keep calpain on ice in an ice bucket in the refrigerator. Activity will be maintained
for 6-12 months under these conditions.
Never freeze calpain.
Do not keep at room temperature for prolonged periods of time.
Calpain can be lyophilized from a 10% sucrose solution and stored dry for
long periods of time.
Calpain Handling
Always maintain neutral pH in the sample. Calpain precipitates at pH<6.0.
Always keep EDTA or EGTA in the calpain sample. Calpain undergoes
autoproteolysis in the presence of Ca2+ and some other cations (Mn2+,
Ba2+, Sr2+, and La3+). Calpain is inactivated
by Zn2+, Co2+, Cu2+, Ni2+, and Fe2+.
Calpain is a cysteine protease. The active site -SH group must be maintained
in the reduced state for activity of the enzyme. Therefore, 2-mercaptoethanol or
dithiothreitol are added to calpain solutions.
Optimum salt concentration for calpain activity is 100-200 mM KCl. Higher
concentrations of salt will inhibit the activity. 1M (NH4)2SO4
inhibits activity by approx 50%.
m-Calpain requires approx 300-800 µM Ca2+ for half-maximal
activity. µ-Calpain requires approx 30-80 µM Ca2+ for half-maximal
activity.
Autolysis of the enzyme begins immediately upon addition of Ca2+.
Incubations of more than 30 min in the presence of Ca2+ at room
temperature will cause complete autolysis of the enzyme. Activity is lost in
proportion to the extent of autolysis.
Inhibitors of calpain include calpastatin (endogenous, specific inhibitor),
chelating agents such as EDTA and EGTA, E-64, calpeptin, leupeptin, and many
others.
Calpastatin can be stored for a short time (< 2 weeks) on ice. For
longer storage, it should be frozen at -80°C. The protein can be
maintained for several months at this temperature. Lyophilized, it can be
stored for even longer periods. Store the samples in aliquots to avoid
freeze-thaw cycles.
Calpastatin Handling
Maximum activity is achieved at pH 7-8, but calpastatin is stable over a wide
range of pH (2-10).
Calpastatin can tolerate a wide range of temperatures also, including
freezing and boiling. Maximum activity is seen at 25-30°C.
Calpastatin requires Ca2+ for binding to calpain, 40-50µM for
µ-calpain and 250-500µM for m-calpain.
Calpastatin is highly susceptible to proteolysis. Inhibitors should be
included in all buffers during purification.
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