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. 2007 Jun 22;3(6):e87. doi: 10.1371/journal.ppat.0030087

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© 2007 Colangeli et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) EMSA assay of radiolabeled P_iniBAC in the presence of Lsr2. Five fmole (0.7 ng) of P_iniBAC were incubated with the following amounts of Lsr2: lane 1, 0 ng; lane 2, 400 ng; lane 3, 200 ng; lane 4, 100 ng; lane 5, 50 ng; lane 6, 25 ng.

(B) Determination of the K_d of Lsr2-binding activity to P_iniBAC. Five fmole (♦) and 50 fmole (○) of radiolabeled P_iniBAC were incubated with the following amounts of Lsr2: 0 ng, 50 ng, 100 ng, 200 ng, 400 ng, and 800 ng; and then analyzed in EMSA assays.

(C) Competition analysis. Fifty fmole of radiolabeled P_iniBAC were incubated with 200 ng of Lsr2 where indicated. Different amounts of either cold P_iniBAC or poly dI-dC were then added as competitors. Lane 1, no Lsr2 (all other lanes contain Lsr2); lane 2, no competitor; lane 3, 7 ng of P_iniBAC; lane 4, 37 ng of P_iniBAC; lane 5, 72 ng of P_iniBAC; lane 6, 7 ng of poly dI-dC; lane 7, 37 ng of poly dI-dC; lane 8, 72 ng of poly dI-dC.

(D) Specificity of DNA binding. Five fmole of radiolabeled P_iniBAC were incubated with 200 ng of Lsr2 where indicated by a “+” followed by treatment with 0.5% SDS or SDS plus 0.1% glutaraldehyde where indicated. Lane 1, radiolabeled P_iniBAC only; lane 2, Lsr2 added; lane 3, SDS only; lane 4, Lsr2 plus SDS; lane 5, SDS plus glutaraldehyde only; lane, 6 Lsr2 plus SDS and glutaraldehyde.