Comparison of protein quantification and extraction methods suitable for E. coli cultures.
Many different extraction and analysis methods exist to determine the protein fraction of microbial cells. For metabolic engineering purposes it is important to have precise and accurate measurements. Therefore six different protein extraction protocols and seven protein quantification methods were tested and compared. Comparison was based on the reliability of the methods and boxplots of the normalized residuals. Some extraction techniques (SDS/chloroform and toluene) should never be used: the measurements are neither precise nor accurate. Bugbuster extraction combined with UV280 quantification gives the best results, followed by the combinations Sonication-UV280 and EasyLyse-UV280. However, if one does not want to use the quantification method UV280, one can opt to use Bugbuster, EasyLyse or sonication extraction combined with any quantification method with exception of the EasyLyse-BCA_P and Sonication-BCA_P combinations.
Ghent University, Laboratory of Industrial Microbiology and Biocatalysis, Department of Biochemical and Microbial Technology, Faculty of Bioscience Engineering, Coupure Links 653, B-9000 Ghent, Belgium. marjan.demey@ughent.be
Marjan
M
De Mey
Gaspard J
GJ
Lequeux
Jo
J
Maertens
Cassandra I
CI
De Muynck
Wim K
WK
Soetaert
Erick J
EJ
Vandamme
D001671:Biochemistry / Q000379:methods*; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D002725:Chloroform / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism*; D006878:Hydroxides / Q000494:pharmacology; D008660:Metabolism; D008828:Microbiological Techniques; D015233:Models, Statistical; D008962:Models, Theoretical; D017680:Potassium Compounds / Q000494:pharmacology; D011506:Proteins / Q000032:analysis*; D011804:Quinolines / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013056:Spectrophotometry, Ultraviolet; D014050:Toluene / Q000494:pharmacology
D006878:Hydroxides; D017680:Potassium Compounds; D011506:Proteins; D011804:Quinolines; D012967:Sodium Dodecyl Sulfate; D014050:Toluene; D002725:Chloroform; C047117:bicinchoninic acid; C029943:potassium hydroxide