( was supplemented with CaCl 2 to a fina...)High-Level Protein Expression, One-Column Purification, and FLAG,,,Epitope Tagging in E. coli

was supplemented with CaCl₂ to a final concentration of 6 mM, then digested at room temperature with a catalytic ratio of thrombin: CBP-FLAG-JNK. To evaluate the extent of digestion over time, the reaction was terminated at various time points (0, 1, and 18 hours). The degree of digestion by thrombin was evaluated by performing SDS-PAGE. 20 l of each time point sample was boiled, then electrophoresed on a 12% Tris-glycine acrylamide gel, and then stained with Coomassie Blue (not shown).

To confirm recognition of FLAG-JNK by anti-FLAG M2 antibody, western blot analysis was performed on the thrombin digest time points. 10-l (~1 g) of sample was subjected to SDS-PAGE, as described above. Samples were electroblotted onto a nitrocellulose filter and detected with anti-FLAG M2 antibody using standard procedures (Figure 4).

Immunoaffinity Pull-Down of c-Jun with FLAG-JNK

It has been shown that transcriptional activity of c-Jun is greatly enhanced in response to phosphorylation.⁷ JNK binds to the c-Jun transactivation domain and phosphorylates Ser-63, Ser-73, and some secondary sites. To demonstrate the utility of a FLAG-fusion protein with the CBP tag removed, we performed an immunoaffinity pull-down of c-Jun from HeLa cell nuclear extract with immobilized FLAG-JNK.

JNK gel was prepared by immobilizing FLAG-JNK on anti-FLAG M2 affinity gel. JNK gel was equilibrated in binding buffer and subsequently incubated with HeLa extract. The affinity gel was pelleted by low-speed centrifugation and washed extensively with binding buffer. The washed gel pellet was boiled in sample buffer then subjected to SDS-PAGE. Duplicate gels were performed. One gel was stained with Coomassie Blue protein stain, the other gel was electroblotte
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