ecl plus  (Bio-Rad)

Journal: Chembiochem

Article Title: Identification of Methylglyoxal Reactive Proteins with Photocaged Glycating Agents

doi: 10.1002/cbic.202500275

Figure Lengend Snippet: A) Structures of the photocaged‐AlkMGO derivatives. B) Western blot read‐out of the glycation of BSA with photocaged AlkMGO derivatives. Chemical‐activated AlkMGO was prepared by hydrolyzing AlkMGO‐DMA with 5% H 2 SO 4 (H 2 O/DMSO, 1/1) at 100 °C for 30 min prior to the labeling step. The crude probe mixture was neutralized with potassium hydroxide to pH 7. BSA (1 mg mL −1 , 90 μL) in PBS (pH 7.4) was incubated with the probe for 1 h. During this period, the samples were either irradiated with UV light (365 nm) or kept in the dark. The incubation with the probe was followed by a CuAAC reaction with biotin‐azide overnight at RT. After separation of the proteins on the SDS‐PAGE, the proteins were transferred to a PVDF membrane and the labeled proteins were visualized with Strp‐HRP and ECL+. For uncropped images, see Figure S7 C, Supporting Information. C) Heat map of the modified peptides identified in BSA that was labeled with AlkMGO‐PC1 trans (250 μM), AlkMGO‐DMA (250 μM, chemically activated prior to labeling) or DMSO for 1 h. D) Venn diagram of the comparison of the total modified residues and the conserved modified residues identified in this study compared to those identified in HSA by Sibbersen et al.

Article Snippet: The biotinylated proteins were visualized with Clarity Western ECL Plus substrate on a ChemiDoc XRS (Bio‐Rad) according to the manufacturer's protocol.

Techniques: Western Blot, Labeling, Incubation, Irradiation, SDS Page, Membrane, Modification, Comparison