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Glutaraldehyde cross-linking

AK Amit Kumar
CC Christopher S. Campomizzi
NJ Natalie Jay
SF Shaun Ferguson
ES Emelie-Jo Scheffler
JL James Lioi
CT Chengjian Tu
JQ Jun Qu
CS Claire Simons
DE D. Fernando Estrada
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Chemical cross-linking of CYP121A1 was carried out using 200 µl aliquots of 30 µM protein in 20 mM HEPES buffer, pH 8.2. CYP121A1 was saturated with econazole for added stability. The cross-linking reaction was initiated by the addition of 10 µl of a freshly prepared solution of 2.3% glutaraldehyde. The reaction was stopped after 20 min by the addition of 80 µl of a 1 M TrisHCl solution. Cross-linked dimers were resolved by electrophoresis on a 5–12% gradient acrylamide gel. Reactions were run in triplicate and subjected to chymotrypsin digest and mass spectrometric analysis as liquid samples. A sample of CYP121A1 that had not been treated with glutaraldehyde was used as a reference.

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