Glycolaldehyde-derived advanced glycation end-products (AGEs) were generated under sterile conditions as we [37 (link),38 (link)] and others [46 (link)] have described previously. Briefly, sterile filtered 30% bovine serum albumin (BSA) solution (Sigma, St. Louis, MO) was incubated with 70 mM glycolaldehyde dimer (Sigma) in sterile PBS without calcium chloride and magnesium chloride for three days at 37°C. Next, the AGE product was dialyzed against sterile PBS for 24 hours at 4°C using gamma-irradiated 10kDa cut-off cassettes (Thermo Scientific, Waltham, MA) to remove unreacted glycolaldehyde dimer. Unmodified control BSA was prepared similarly, without the addition of glycolaldehyde dimer. Protein concentration was determined by BCA assay (Thermo Scientific), and the extent of BSA modification was confirmed by fluorescence, absorbance, and loss of primary amines [46 (link)–49 (link)].
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Glycolaldehyde-Derived Advanced Glycation Endproducts
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Corresponding Organization : Purdue University West Lafayette
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- Incubation of sterile filtered 30% bovine serum albumin (BSA) solution with 70 mM glycolaldehyde dimer in sterile PBS without calcium chloride and magnesium chloride for three days at 37°C
- Extent of BSA modification, confirmed by fluorescence, absorbance, and loss of primary amines
- Preparation of unmodified control BSA similarly, without the addition of glycolaldehyde dimer
- Positive control: Incubation of BSA with glycolaldehyde dimer
- Negative control: Unmodified BSA without glycolaldehyde dimer
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