Protein extraction from activated sludge.
Two methods for the separation of protein originating from activated sludge were compared. In one method, the total protein was isolated out of the activated sludge (crude extract). These samples included all dissolved proteins originating from the bacterial cells and biofilm made up of extracellular polymeric substances (EPS). Every time polyacrylamide gel electrophoresis (PAGE) was done, the protein bands from samples of crude extract were covered by polymeric substances including carbohydrates, uronic acids or humic compounds. Using the immunoblot technique it was possible to demonstrate the presence of the heat shock protein HSP70 in crude extracts of activated sludge. The comparison of protein fingerprints required that clear and distinct bands appear on the PAGE analysis. To this end, a procedure to separates bacterial cells from the EPS was developed. Bacterial cells were separated by incubation with EDTA and subsequent filtration. The isolated cells were directly incubated in a sample buffer.
Denecke
M
M
Department of Waste Management and Urban Water Management, Faculty of Civil Engineering, University of Duisburg-Essen, Universitätsstrasse 15, 45141 Essen, Germany. martin.denecke@uni-essen.de
D001426:Bacterial Proteins / Q000302:isolation & purification*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D018840:HSP70 Heat-Shock Proteins / Q000302:isolation & purification; D012722:Sewage / Q000737:chemistry* / Q000382:microbiology
D001426:Bacterial Proteins; D018840:HSP70 Heat-Shock Proteins; D012722:Sewage