A Standard Operating Procedure (SOP) for the preparation of intra- and extracellular proteins of Clostridium acetobutylicum for proteome analysis.
We report on the development of a Standard Operating Procedure (SOP) for extraction and handling of intra- and extracellular protein fractions of Clostridium acetobutylicum ATCC 824 for reproducible high quality two-dimensional gel electrophoresis (2-DE) analyses. Standardized cells from a phosphate-limited chemostat were used to evaluate different protein preparation methods. For the preparation of the secretome, a dialysis/ultrafiltration procedure resulted in higher protein yields and proved to be more reliable compared to different precipitation methods using TCA, DOC-TCA, acetone, and PEG 6000. Sonication was found to be the most efficient method among different tested techniques of cell disruption for the analysis of the intracellular proteome. Furthermore, the effect of protease inhibitors and sample storage conditions were tested for both intra- and extracellular protein samples. Significant changes in the protein pattern were observed depending on the addition of protease inhibitors. 2-DE gels with a pH gradient from 4 to 7 prepared according to the developed SOP contained at least 736 intracellular and 324 extracellular protein spots.
Schwarz
Katrin
K
University of Rostock, Institute of Biological Sciences, Division of Microbiology, Rostock, Germany.
Fiedler
Tomas
T
Fischer
Ralf-Jörg
RJ
Bahl
Hubert
H
D001426:Bacterial Proteins / Q000302:isolation & purification*; D002458:Cell Fractionation / Q000379:methods; D011232:Chemical Precipitation; D046969:Clostridium acetobutylicum / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D040901:Proteomics / Q000379:methods*; D013010:Sonication; D014462:Ultrafiltration
D001426:Bacterial Proteins