TY - JOUR
T1 - Purification of Cap Z from Chicken Skeletal Muscle
AU - Casella, James F.
AU - Cooper, John A.
PY - 1991/1
Y1 - 1991/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0025764370&partnerID=8YFLogxK
U2 - 10.1016/0076-6879(91)96015-J
DO - 10.1016/0076-6879(91)96015-J
M3 - Article
C2 - 1851938
AN - SCOPUS:0025764370
SN - 0076-6879
VL - 196
SP - 140
EP - 154
JO - Methods in enzymology
JF - Methods in enzymology
IS - C
ER -