Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

188 Brokx and Vogel
Fig. 2. 20% SDS-PAGE gel of calmodulin and its proteolytic fragments. Lane S:
protein standards (molecular weight indicated in kDa), lane 1: calmodulin, lane 2:
TR1C, lane 3: TR2C, lane 4: TM1, lane 5: TM2.
3. Add 200 mL of the trypsin stock (to 250 mg/mL trypsin) and incubate for 40 min
at 37°C.
4. After the 40 min, add 400 mL of the soybean trypsin inhibitor stock (to 500 mg/mL
STI), and cool the mixture on ice. Take a sample for SDS-PAGE (see Fig. 2).
5. Quickly apply the digest to the G50 column by the following procedure:
a. First, disconnect the column from the pump and let the excess buffer run into
the top of the column until only a very tiny amount of buffer covers the top of
the matrix.
b. Then very gently apply the digest solution and let it run into the column in the
same manner.
c. Repeat this again a few times with small amounts (1 mL) of buffer A until the
sample is washed completely into the column.
d. Then gently apply some buffer A on top of the column and reconnect the
column to the pump. Run the column with buffer A at approx 0.35 mL/min.
Collect 15 min fractions.
6. Measure A 280nm of the column fractions. There will basically be two peaks; the
first will be undigested protein and trypsin as well as its inhibitor; the second
(about 50 mL later) will be the two tryptic fragments, TR1C and TR2C. Collect
the second peak and assess purity by SDS-PAGE (see Fig. 2).
7. Equilibrate a phenyl-Sepharose column with buffer B. Run at 2 mL/min for
approx 2 h. The phenyl-Sepharose matrix is very stable; it may be quickly equilibrated
by removing the storage solution and then resuspending in buffer B.
8. Apply the pooled fractions onto the phenyl-Sepharose column and run at approx
2 mL/min. CaM and its tryptic fragments will bind to this matrix due to the