Figure 3. Products from the translocational assay using native TIGR: demonstration of pausing with the HincII truncation

The products are shown on a 15% SDS/PAGE gel, with markers indicating band size in kilodaltons (kDa). A. Full length construct. An additional upper band appears in lane 1 (complete translation products, no PK treatment) but disappears in lanes 2, 3, and 4 (lanes have been treated with PK). This band is completely PK sensitive and probably represents product not associated with the ER membrane. B. AvrII truncated construct. Insignificant faint bands such as that seen after detergent and PK treatment (lane 4) occasionally occur; this band does not correspond with the size of the observed translation products in lanes 1, 2, and 3. C. HincII truncated construct. Lanes 1, 2, 3, and 4 are the same for all constructs: Lane 1, no proteinase K (-PK) treatment; Lane 2, proteinase K treatment (+PK); Lane 3, treatment with EDTA, then proteinase K (E+PK); Lane 4,treatment with detergent and PK (D+PK).