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A project‐based laboratory using 2‐D protein gel electrophoresis and purification and characterization of glutamine synthetase
Author(s) -
Hemmingsen Jens,
Flick Hollie,
Couden Scott
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a976-c
Subject(s) - glutamine synthetase , glutamine , gel electrophoresis , enzyme , polyacrylamide gel electrophoresis , biochemistry , chromatography , ammonium , chemistry , microbiology and biotechnology , two dimensional gel electrophoresis , electrophoresis , proteomics , biology , amino acid , gene , organic chemistry
A project‐based undergraduate laboratory has been developed and implemented to explore nitrogen metabolism in E. coli . Cells are grown on minimal media containing either glutamine or ammonium chloride as the only nitrogen source. Cleared cell lysates from the two cultures are analyzed by two‐dimensional (2‐D) protein gel electrophoresis to determine differences in protein expression. Cells grown on glutamine show increased expression of the enzyme glutamine synthetase due to activation of the glnALG operon. In a subsequent experiment, students purify E. coli glutamine synthetase using reversible Zn 2+ precipitation of the enzyme and affinity chromatography. The purified enzyme is characterized using gel electrophoresis, BCA protein determination and gamma‐glutamyl transferase activity assays. Experiments can be completed in four class periods with minimal work required between classes. The laboratory provides students with a wide variety of experimental techniques and some exposure to proteomics methods through 2‐D gel electrophoresis.