Protein Protocols Protein Protocols

88 Akins and Tuan
an active form after removal of SDS (8,9); however, this method is inconvenient and
potentially unreliable. A preferred method for determining native protein activity after
electrophoresis involves the use of nonionic detergents like Triton X 100 (Tx-100)
(10); however, proteins do not separate based on molecular size. The assignment of M r
in the nonionic Tx-100 system requires the determination of mobilities at several different
gel concentrations and “Ferguson analysis” (11–13). The CAT gel system combines
the most useful aspects of the SDS and Tx-100 systems by allowing the separation
of proteins based on M r with the retention of native activity.
Previous studies have described the use of CTAB and the related detergent
tetradecyltrimethylammonium bromide (TTAB), in electrophoretic procedures for the
determination of M r (14–18). In addition, as early as 1965, it was noted that certain
proteins retained significant levels of enzymatic activity after solubilization in CTAB
(19). A more recent report further demonstrated that some proteins even retained enzymatic
activity after electrophoretic separation in CTAB (14). Based on the observed
characteristics of CTAB and CTAB-based gel systems, we developed the CAT gel system.
In contrast to previous CTAB-based gel methods, the CAT system is discontinuous
and allows proteins to be “stacked” prior to separation (see refs. 6 and 7). CAT gel
electrophoresis is a generally useful method for the separation of proteins with the
retention of native activity. It is also an excellent alternative to SDS-based systems for
the assignment of protein M r (see Note 1).
1.2. Basic Principles of CAT Gel Operation
The CAT gel system is comprised of two gel matrices and several buffer components
in sequence. A diagram of the CAT gel system is shown in Fig. 1. In an applied
electric field, the positive charge of the CTAB–protein complexes causes them to
migrate toward the negatively charged cathode at the bottom of the system. The arginine
component of the tank buffer also migrates toward the cathode; however, arginine is a
zwitterion, and its net charge is a function of pH. The arginine is positively charged at
the pH values used in the tank buffer, but the pH values of the stacking gel and sample
buffer are closer to the pI of arginine, and the arginine will have a correspondingly
lower net positive charge as it migrates from the tank buffer into these areas. Therefore,
the interface zone between the upper tank buffer and the stacking gel/sample buffer
contains a region of high electric field strength where the sodium ions in the stacking
gel/sample buffer (Tricine-NaOH) move ahead of the reduced mobility arginine ions
(Tricine-arginine). In order to carry the electric current, the CTAB-coated proteins
migrate more quickly in this interface zone than in the sodium-containing zone just below.
As the interface advances, the proteins “stack,” because the trailing edge of the applied
sample catches up with the leading edge. When the cathodically migrating interface
zone reaches the separating gel, the arginine once again becomes highly charged owing
to a drop in the pH relative to the stacking gel. Because of the sieving action of the
matrix, the compressed bands of stacked proteins differentially migrate through the
separating gel based on size.
Two features of CTAB-based gels set them apart from standard SDS-based electrophoretic
methods. First, proteins separated in CTAB gels migrate as a function of log
M r across a much broader range of molecular weights than do proteins separated in