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. 2010 Dec 17;193(4):909–917. doi: 10.1128/JB.01175-10

FIG. 5.

FIG. 5.

His6-NagR binds to the nag operon promoter. (A) His6-NagR was purified using a HisTrap nickel column, separated on a 12% SDS-PAGE gel, and visualized after staining with Coomassie blue. The lanes contained desalted NagR used for EMSA (NagR), molecular weight ladder (L), cell lysate (Lys), pellet obtained after ultracentrifugation of lysate (P), soluble crude extract (Sup), column flowthrough after soluble cell extract addition (Ft), wash with buffer B containing 200 mM imidazole (W1), and elution of His6-NagR with buffer B containing 400 mM imidazole (W2). (B) EMSA analysis of His6-NagR binding to the nag operon promoter region. The lanes contain the following: 32P-labeled nag operon promoter probe (probe); 32P-labeled nag operon promoter probe with 250 nM His6-NagR and 250 μM GlcNAc (NagR); 32P-labeled nag operon promoter probe with 250 nM His6-NagR, 250 μM GlcNAc, and a 20-fold molar excess of unlabeled probe (specific); and 32P-labeled nag operon promoter probe with 250 nM His6-NagR, 250 μM GlcNAc, and a 20-fold molar excess of unlabeled nonspecific DNA competitor (nonspecific).