With the aim of identifying complexes formed by the above-listed
proteins, we subjected GMPs to a cross-linking treatment, followed by
2D-electrophoretic separation (see Materials and methods). For crosslinking,
we used FA due to three important properties [38]. First, FA possesses
the shortest possible spacer arm, forming cross-bridges between
molecules that are necessarily in very close proximity (2.3–2.7 Å), a
characteristic of protein complexes [38]. Second, the cross-linking reaction
rate of FA is very high, enabling it to capture short-lived protein
complexes. Third, FA-mediated cross-linking is reversible. In 2D-PAGE,
proteins that are not part of a complex created a diagonal line, as seen
in Fig. 6A. However, proteins held together by FA cross-linking bridges
were separated only in the second PAGE, and, therefore, appear in
Fig. 6A as bands below the diagonal line.