(A) Cross-linking of endogenous HeLa cell lysate proteins with fluorescent Akt1 substrate peptide 2 in the presence of different cross-linkers OPA or aldehydes 11-14. HeLa cell lysates and fluorescent Akt1 substrate peptide 2 were treated with cross-linkers OPA or 11-14 for 20 min at r.t., followed by SDS-PAGE and in gel scanning fluorescence. (B) Synthesis of the thiophene-2,3-dialdehyde based cross-linker 17. (C) Cross-linking of Akt1 and fluorescent substrate peptide 2 with cross-linker 17. Akt1 and fluorescent peptide 2 were treated with cross-linker 17, for 20 min at r.t., followed by SDS-PAGE and in gel scanning fluorescence. (D) Cross-linking of Akt1 and fluorescent peptide substrate 2 with cross-linker 17 in the presence of increasing amounts of HeLa cell lysate proteins. Akt1 and substrate peptide 2 were treated with cross-linker 17 for 20 min at r.t., followed by SDS-PAGE and in gel scanning fluorescence.