Zymographic Techniques for the Analysis of Bacterial Cell Wall in Bacillus.
Zymography of cell wall hydrolases is a simple technique to specifically detect cell wall or peptidoglycan hydrolytic activity. The zymographic method can be used for assessing the hydrolytic activities of purified target proteins, cell surface proteins, and proteins secreted to culture. Here, methods of cell wall and peptidoglycan purification, extraction of cell surface proteins containing cell wall hydrolases, and zymographic analysis are described. The purified or extracted proteins are separated by electrophoresis using an SDS gel containing cell wall or peptidoglycan material and then the proteins are renatured in the gel. The renatured cell wall hydrolases in the gel hydrolyze the material around the proteins. The cell wall or peptidoglycan in the gel is stained by methylene blue and the hydrolyzed material cannot be stained, resulting in the detection of cell wall hydrolytic activities of the enzymes on the gel.
Division of Gene Research, Department of Life Sciences, Research Center for Human and Environmental Sciences, Shinshu University, 3-15-1 Tokida, Ueda, Nagano, 386-8567, Japan. tatsuf@shinshu-u.ac.jp.
Tatsuya
T
Fukushima
Department of Applied Biology, Graduate School of Science and Technology, Shinshu University, 3-15-1 Tokida, Ueda-shi, Nagano, 386-8567, Japan. jsekigu@shinshu-u.ac.jp.
Junichi
J
Sekiguchi
D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000378:metabolism; D002473:Cell Wall / Q000201:enzymology; D006867:Hydrolases / Q000302:isolation & purification / Q000378:metabolism; D006868:Hydrolysis; D010457:Peptidoglycan / Q000302:isolation & purification / Q000378:metabolism*
D001426:Bacterial Proteins; D010457:Peptidoglycan; D006867:Hydrolases
Carboxypeptidase; Endopeptidase; Glucosaminidase; Gram-positive bacteria; Lipoteichoic acid; Lytic transglycosylase; Muramidase; Wall teichoic acid