Journal: ACS Bio & Med Chem Au

Article Title: A Click Chemistry-Based Biorthogonal Approach for the Detection and Identification of Protein Lysine Malonylation for Osteoarthritis Research

doi: 10.1021/acsbiomedchemau.4c00151

Figure Lengend Snippet: Assessment of the ability of MA-diyne to metabolically label proteins. (A) Mouse primary chondrocytes were incubated with the indicated concentration of MA-diyne for 6 h. The cell lysates were then clicked with IR680-azide followed by in-gel fluorescence analysis. (B) Mouse primary chondrocytes were incubated with 100 μM MA-diyne for the indicated time points. Cell lysates were then clicked with IR680-azide and in-gel fluorescence analyses. β actin was used as a loading control. (C) Confocal microscopic fluorescence image depicting the ready uptake of the MA-diyne into the cells and subcellular localization of malonylated proteins in the primary chondrocytes. The primary chondrocytes grown on coverslips were labeled with 100 μM MA-diyne for 6 h and then subjected to click chemistry with Carboxyrhodamine 110 Azide (fluorescein isothiocyanate-FITC tag) after fixing and permeabilization. Scale bar: 50 μM. (D) Box plot showing the concentration of intracellular malonyl-CoA in the cells after incubation with different concentrations of MA-diyne for 4 h. n = 4. Data are presented as mean ± SEM. Three group comparisons were evaluated using two-way ANOVA. Significance is noted as ns p > 0.05, * p < 0.05, and ** p < 0.01.

Article Snippet: Briefly, a click reaction master mixture for the required volume of protein lysates was prepared by mixing the reagents in the following order: 100 μM IR680 azide dye (click chemistry tools) for in-gel fluorescence/100 μM Biotin azide (Click chemistry tools) for streptavidin enrichment and Western blotting, 100 μM tris ((1-benzyl-4-triazolyl) methyl) amine (TBTA) (Click chemistry tools), 1 mM tris (carboxyethyl) phosphine (TCEP) (Sigma), and 1 mM CuSO 4 .

Techniques: Metabolic Labelling, Incubation, Concentration Assay, Fluorescence, Control, Labeling

Journal: ACS Bio & Med Chem Au

Article Title: A Click Chemistry-Based Biorthogonal Approach for the Detection and Identification of Protein Lysine Malonylation for Osteoarthritis Research

doi: 10.1021/acsbiomedchemau.4c00151

Figure Lengend Snippet: Pulse-chase experiment to determine the dynamic nature of lysine malonylation. Wild-type mouse primary chondrocytes (A) and Sirt5 KD primary chondrocytes (B) were labeled with 200 μM MA-diyne for 1 h and then pulse chased with 200 μM Meldrum acid (precursor). The lysates were collected at the indicated time points followed by a click reaction with IR680-azide and in gel fluorescence analysis. β actin was used as a loading control. The same blot was probed with an anti-Sirt5 antibody to quantify the knockdown efficiency.

Article Snippet: Briefly, a click reaction master mixture for the required volume of protein lysates was prepared by mixing the reagents in the following order: 100 μM IR680 azide dye (click chemistry tools) for in-gel fluorescence/100 μM Biotin azide (Click chemistry tools) for streptavidin enrichment and Western blotting, 100 μM tris ((1-benzyl-4-triazolyl) methyl) amine (TBTA) (Click chemistry tools), 1 mM tris (carboxyethyl) phosphine (TCEP) (Sigma), and 1 mM CuSO 4 .

Techniques: Pulse Chase, Labeling, Fluorescence, Control, Knockdown