Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

330 Kobayashi
Fig. 2. Tricine/SDS/PAGE (15) of the proteins obtained from bovine lung extract
by Amlexanox- and Cromolyn- affinity chromatographs. The gels were stained with
Coomassie brilliant blue R–250. The numbers on the left indicate molecular mass standards
(Bio-Rad) in kilo-Daltons. Affinity columns were eluted with an EGTA-containing
buffer (Buffer C). Lane 1, acetylated amino-Toyopearl column for control; lane 2,
Amlexanox-Toyopearl column; lane 3, Cromolyn-Toyopearl column.
3. Elute the protein with 150 mL of Buffer C (20 mM Tris-HCl, 2 mM EGTA,
pH 7.5) and then with Buffer D. Monitor the column eluate at 280 nm, and collect
the fractions corresponding to each major peak.
4. To verify purity of the fraction, subject the column eluate to Tricine/SDS/PAGE
(15), Western-blotting, and reverse phase (RP)-HPLC.
3.7.3. Identification and Separation of Ca 2+ -Binding Proteins
from a Drug-Affinity Column by RP-HPLC (Fig. 3)
In most instances, the identification and separation of each Ca 2+ -binding
proteins (EF-hand proteins) can be archived by analytical RP-HPLC combined
with SDS/PAGE. Analytical RP-HPLC should be carried out on a narrow C 18
column using a 0–60% acetonitrile gradient. Analysis on a Tricine/SDS/PAGE
as reported by Schägger and von Jagow (15) is often helpful in confirming the
homogeneity of the preparation.
1. The protein sample obtained from the drug-affinity column could be boiled in a
water bath for 3 min to eliminate heat labile proteins. Calmodulin and other
EF-hand proteins such as S-100 protein family and calcyphosine are heat stable.
Alternatively, protein sample from a drug-affinity column could be separated
using an ion-exchange column chromatography, such as Q-Sepharose, DEAE
cellulose, and Mono Q-FPLC.