Figure 1
Principle of the 4 F strategy. T: target protein, L: ligand. Without staining, the portion of the gel corresponding to molecular weights above that of a single albumin molecule (70 kDa) was excised in six slices using the protein markers as a guide. The slices corresponded to MWs of 70–100 kDa, 100–130 kDa, 130–180 kDa, 180–250 kDa, 250–300 kDa, and greater than 300 kDa. 1. The target protein has to be identified in the same slice as the ligands. 2. The molecular weight of each complex is the sum of the theoretical MWs of the target and the ligand. a) Ligands are not identified in the control, as observed for L1 and L3. b) Target complexes bind to the beads nonspecifically after cross-linking. To be conservative, the ligand should be identified at a MW higher than that of the control (> = 300 kDa) and outside of the neighboring range (250–300 kDa), as for L2. (L2-T-L3 is an example of a complex with interactions other than binary interactions). 3. Proteins identified in the same range as in the control group or in a neighboring range are eliminated as contaminants and are not shown in the figure.