Isolation and solubilization of gram-positive bacterial cell wall-associated proteins.
This chapter describes a simple, rapid and reproducible method to prepare bacterial cell wall extracts for two-dimensional gel electrophoresis (2DE). The extraction process uses mutanolysin, an N-acetylmuramidase, to gently solubilize cell wall-associated proteins from Gram-positive prokaryotes. The cells are first washed with buffer and resuspended in a solution containing mutanolysin. Following incubation at 37 degrees C, the sample is centrifuged and the supernatant containing the soluble cell wall-associated proteins is harvested. Following a brief precipitation step, the pellet is solubilized in sample buffer ready for isoelectric focusing and 2DE analysis.
Cole
Jason N
JN
University of Wollongong, Wollongong, Australia.
Djordjevic
Steven P
SP
Walker
Mark J
MJ
D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002473:Cell Wall / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional; D012995:Solubility; D013297:Streptococcus pyogenes / Q000378:metabolism*
D001426:Bacterial Proteins