Numerous actin-binding proteins interact specifically with the ends of actin filaments. These proteins fall into two groups: those that “cap” the ends of actin filaments and inhibit actin monomer addition and subtraction at that end, versus those that both cap and sever actin filaments. Cap Z is a member of the first group, referred to as capping proteins, whereas gelsolin, severin, fragmin, and villin are members of the second. Cap Z is a heterodimeric protein and cDNAs encoding reveals two subunits of Cap Z predict proteins with maximal sizes of 33,960 and 31,352 Da, respectively, for the α and β subunits of Cap Z. This chapter presents two protocols on purification procedures of Cap Z. The first is essentially as described in the original purification of Cap Z. This procedure has the advantage that it can be performed in most laboratories with readily available equipment. The second protocol has the advantage of being more rapid; however, it requires the capability for high-performance liquid chromatography or fast protein liquid chromatography (FPLC) and the use of one specialized column (a 1.6 × l0 cm Mono S column). This procedure may also result in higher yields. Both procedures should yield milligram quantities of Cap Z. It is suggested that the activity of Cap Z be followed throughout the purification procedure and that the various stages of purification be analyzed on 10% SDS-polyacrylamide gels as well. However, as familiarity with the procedure is gained, the purification can be carried out by monitoring with gels or activities alone.
ASJC Scopus subject areas
- Molecular Biology