218
- Exchange the protein into the Working Buffer and concen-
trate to around 10 μM. - Set up four 20 μL reactions in 1.8 mL microcentrifuge tubes
to contain 5 μM protein (10 μL), 10× Working Buffer (2 μL)
and the following reagents:
(a) 1 mM EGTA (2 μL of 10 mM stock).
(b) 1 mM CaCl 2 (2 μL of 10 mM stock).
(c) 1 mM EGTA (2 μL) + 25 μM inhibitor (1μL of 1 mM
stock).
(d) 1 mM CaCl 2 (2 μL) + 25 μM inhibitor (1 μL). - Add ddH 2 O to a total volume of 20 μL.
- Incubate the reactions for 20 min at 37 °C with or without
shaking. - Stop the reaction by adding 11 μL SDS-Sample Buffer.
- Heat samples for 10 min at 70 °C or for 3 min at 90 °C.
- Load each sample onto a 10 %, 10-lane, SDS-PAGE gel and
run for 50 min in MOPS buffer. - Analyze the results (Fig. 3 ).
4 Notes
- An arginine residue in the thrombin cleavage site of the
pET28a+ vector was mutated to glycine for the expression of
recombinant and active TbMCA2. This mutant was made
after it was realized that the protein linker in this region was
being cleaved by the enzyme and adversely removing the His-
tag and may be something to consider when working with
arginine-specifi c peptidases in general.
3.5.7 Inhibitor Binding
Gel-Shift Assays
Fig. 3 TbMCA2 gel-shift assay. Inhibitor Z-VRPR-FMK binds to active TbMCA2 on
the addition of Ca 2+ (as indicated by a small increase in apparent molecular
weight in Lanes 1 , 3 and 5 ) but not in the presence of EGTA ( Lane 4 ). Z-VRPR-
FMK also protects active TbMCA2 from autoproteolysis in the presence of Ca 2+
(autoproteolysis shown in Lane 6 )Karen McLuskey et al.http://www.ebook3000.com