Proteins as Distance Layer

Reactive proteins are cross-linked by exposure to the UV-light of a Stratagene DNA cross-linker at 25 mJ for 5 minutes. The UV-light activates aromatic amino-acids (Tyr, 

Phe) and thiols on the outside of the protein, which leads to coupling to the slide surface as well as crosslinking between proteins. 

Many standard proteins lacking sufficient aromatic amino acids and thiols on their surface as e.g. urease need reactive photo cross-linking. For that purpose the protein stock solutions 5% (w/v in distilled water) is mixed with a solution of 3% (w/v) 4,4 -diazidostilbene-2,2 disulfonic acid disodiumsalt tetrahydrate (DIAS) in a ratio of 10 1 (v / v). The mixture is spin-coated onto the mirror and activated with a monolayer l-(3-aminopropyl)-methyl-diethoxysilane. Amino-silane, an efficient adhesion promoter, is applied via vapor silanization as described above. Finally the spin-coated protein film is cross-linking via irradiation with UV-light for 30 seconds (350 nm, 60 W), The application of wavelength less than 350 nm will lead to considerable DIAS decomposition, therefore DNA cross-linker devices as used above are not recommended for this method. 

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