Figure 3. Purification of cross-linked... | Rockefeller University Press

Figure 3.

$Figure 3. Purification of cross-linked SecA–SecYEG complexes. Samples of purified SecYEG, SecA, and their cross-linked products were analyzed by (A and B) SDS-PAGE: A794–Y268EG (SecAY794C cross-linked to SecYK268CEG), and similarly, A795–Y268EG, A794–Y183EG, and A795–Y183EG. The SecYEG and SecA samples were loaded purely as markers and the quantities loaded have no relation to the amounts used in the cross-linking experiment (see Materials and methods for further details). Molecular weight standards are shown on the left side of each panel. (C) Size exclusion chromatography: SecY183CEG (YEG), SecA794C (A) and A795–Y268EG (A-YEG), and a dimer of SecYEG produced by a genetic fusion of two SecY subunits (Y-Y; Duong, 2003). See Fig. S2 for analysis of the eluting A795-Y268EG fractions by SDS-PAGE. Again, the SecYEG, SecA, and SecY-Y samples were loaded in order to calibrate the column and their quantities have no relation to the cross-linked samples. Note that the mutant forms of SecA and SecYEG used elute from the column like their wild-type counterparts.$

Purification of cross-linked SecA–SecYEG complexes. Samples of purified SecYEG, SecA, and their cross-linked products were analyzed by (A and B) SDS-PAGE: A₇₉₄–Y₂₆₈EG (SecA_Y794C cross-linked to SecY_K268CEG), and similarly, A₇₉₅–Y₂₆₈EG, A₇₉₄–Y₁₈₃EG, and A₇₉₅–Y₁₈₃EG. The SecYEG and SecA samples were loaded purely as markers and the quantities loaded have no relation to the amounts used in the cross-linking experiment (see Materials and methods for further details). Molecular weight standards are shown on the left side of each panel. (C) Size exclusion chromatography: SecY_183CEG (YEG), SecA_794C (A) and A₇₉₅–Y₂₆₈EG (A-YEG), and a dimer of SecYEG produced by a genetic fusion of two SecY subunits (Y-Y; Duong, 2003). See Fig. S2 for analysis of the eluting A₇₉₅-Y₂₆₈EG fractions by SDS-PAGE. Again, the SecYEG, SecA, and SecY-Y samples were loaded in order to calibrate the column and their quantities have no relation to the cross-linked samples. Note that the mutant forms of SecA and SecYEG used elute from the column like their wild-type counterparts.