反应详情

温度

37 °C

[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⟧

1. customproduction (see Results, below)
2. customapproximately 40 min
3. additionwas added to a final concentration of 50 μg/ml
4. customC-subunit production (see Results, below)
5. customLysates were prepared
6. customCell debris was removed by centrifugation and 15 μl aliquots of the supernatant
7. customFor Western blot analyses, 50 μg of reduced and denatured lysate proteins, prepared from unlabeled E