Figure 9.

Visualization of the high-molecular-weight complexes by 2D gels. Ag8(8) or Ag8.653 cells (6 × 10⁶) were metabolically labeled overnight and then treated with 150 μg/ml DSP. Protein complexes were immunoprecipitated with protein A-Sepharose alone for Ag8(8) or anti-BiP and protein A-Sepharose for Ag8.653. The samples were first electrophoresed under nonreducing conditions to separate different cross-linked complexes that might be present (1). The gel strip corresponding to a single sample was cut from the first gel and equilibrated in 5 ml of reducing SDS sample buffer for 40 min at room temperature on a rocker to reduce DSP and liberate the various proteins in the complex. The gel strip was then placed on the top of a second gel and run at a 90° angle to the first (2). After staining the gel was dried and a film exposed. A and B is obtained from Ag(8) and Ag8.653 cells, respectively.