2-Mercaptoethanol → Glycylglycine

Participants

Reaction Conditions

Reaction Steps

[3H]Fatty acid labeling of PK-A C-subunit produced in E. coli. Four ml cultures of the double transformants were shaken at 37° C. to an OD600 of 0.5 in LB broth+100 μg/ml ampicillin and 100 μg/ml kanamycin sulfate. Isopropyl-β-D-thiogalactopyranoside (IPTG) was then added to a final concentration of 1 mM to induce NMT production (see Results, below). When the cultures reached OD600 =1.0 (approximately 40 min later), nalidixic acid was added to a final concentration of 50 μg/ml to induce C-subunit production (see Results, below). [3H]Myristate (New England Nuclear; 39.3 Ci/mmol, 113 μCi added per ml of culture), [3H]palmitate (New England Nuclear; 30 Ci/mmol, 143 μCi/ml), or [3H]10-(propoxy)decanoate [Heuckeroth and Gordon, Proc. Natl. Acad. Sci. USA 86, 5262-5266 (1989)] (31.7 Ci/mmol, 800 μCi/ml) was added simultaneously with the nalidixic acid. Cultures were shaken for an additional 20 min at 37° C. and the cells were harvested by centrifugation. Lysates were prepared by boiling E. coli contained in the pellet for 10 min in 40 μL of a solution of 125 mM Tris, pH 8.0, 4% SDS, 20% glycerol, 10% β-mercaptoethanol, and 0.2M dithiothreitol. Cell debris was removed by centrifugation and 15 μl aliquots of the supernatant were subjected to SDS-PAGE [Laemmli, Nature 227, 680-685 (1970)] and subsequent fluorography using EN3HANCE (New England Nuclear) autoradiography enhancer. For Western blot analyses, 50 μg of reduced and denatured lysate proteins, prepared from unlabeled E. coli producing Gly2 -C-subunit or Ala2 -C-subunit, were separated by SDS-PAGE, electroblotted onto nitrocellulose [Burnette, Anal. Biochem. 112, 195-230 (1981)], and the filters probed with polyclonal, monospecific rabbit antisera raised against purified mouse C-subunit. Antigen-antibody complexes were visualized with 125I-protein A [Burnette, supra⟧

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