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. 2015 Jun 19;197(14):2432–2441. doi: 10.1128/JB.00186-15

FIG 3.

FIG 3

Ten percent SDS-PAGE gels of the five purified enzymes and immunoblot analyses of the five purified proteins. Lane 2 contained approximately 30 μg of purified protein. For immunoblot analyses (lanes 3 and 4), 30 μg cell extract separated by 10% SDS-PAGE was blotted onto a cellulose-nitrate membrane, and blots were incubated with the respective antibodies. Lane 1 in each panel is a 6-μl PageRuler plus prestained protein ladder (Thermo Scientific, USA). (A) Lane 2, purified kustc0457/58; lane 3, incubation with the preimmune serum of anti-kustc0457/58; lane 4, incubation with anti-kustc0457/58. (B) Lane 2, purified HDH (kustc0694); lane 3, incubation with the preimmune serum of anti-HDH; lane 4, incubation with anti-HDH. (C) Lane 2, purified HOX (kustc1061); lane 3, incubation with affinity-purified anti-HOX. (D) Lane 2, purified NXR (kustd1700/03/04); lane 3, incubation with affinity-purified anti-NXR. (E) Lane 2, purified HZS (kuste2859-61); lane 3, incubation with affinity-purified anti-HZS. (F) Lane 2, incubation with only secondary antibody. Asterisks indicate expected target subunits. It should be noted that the dominant heme-containing proteins of K. stuttgartiensis were clearly visible as yellowish (light gray in this figure) bands on the blots; therefore, they were distinguishable from a positive immunoblot reaction.