alkyne azdye 647  (Click Chemistry Tools)

Journal: Science Advances

Article Title: Mechanical loading induces the longitudinal growth of muscle fibers via a rapamycin-insensitive mechanism

doi: 10.1126/sciadv.aec5134

Figure Lengend Snippet: ( A ) Illustration of how an azide-bearing analog of methionine, called ANL, can get incorporated into NSPs in mice that express a mutated form of the methionyl-tRNA synthetase (MetRS L274G ). Once incorporated, a click reaction with a fluorophore bearing a reactive DBCO or alkyne group can be used to label the azide on the ANL. ( B ) Genotyping (polymerase chain reaction) and Western blot (WB) analysis of tails from the following: (i) floxed STOP GFP-2A-MetRS L274G transgenic mice, (ii) mice that express CMV-Cre plus floxed STOP GFP-2A-MetRS L274G , and (iii) MetRS L274G+/+ mice that ubiquitously coexpress GFP-2A-MetRS L274G in the absence of CMV-Cre. ( C to F ) MetRS L274G+/+ mice were subjected to an MOV or sham (control) surgery. After 7 days, the mice were injected with ANL or the vehicle (PBS), and the plantaris muscles were collected 24 hours later. (C) Representative in-gel fluorescence images showing the DBCO-Cy5.5–labeled NSPs and total protein levels in each sample. (D) Quantification of the NSP to total protein ratio expressed relative to the mean of the control samples. (E) Representative cross section showing Alkyne-AZDye 647–labeled NSPs and total protein from a pair of plantaris muscles (control and MOV) that were visualized with fluorescence microscopy. Scale bar, 500 μm. (F) NSP to total protein ratio for each sample was expressed relative to the mean of the control samples. For illustrative purposes, the signals for Cy5.5 and AZDye 647 were pseudo-colored green. Values in the bars are presented as the group means ± SEM. n = 4 muscles per group. The data were analyzed with a Student’s t test (D) or a paired t test (F). * denotes significantly different from control, P < 0.05.

Article Snippet: Specifically, a 300 μl of reaction mixture (enough for six sections) was prepared by adding the following reagents in the order listed along with vortexing between each addition: (i) 277.5 μl of PBS, (ii) 1.5 μl of 20 mM Tris [(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine in DMSO, (iii) 3 μl of 100 mM CuSO4 in diH 2 O, (iv) 18 μl of 50 μM alkyne-AZDye 647 (Click Chemistry Tools #CCT-1301) in DMSO or 6 μl of 500 μM alkyne-AZDye 555 (Click Chemistry Tools #CCT-1289) in DMSO, and (v) 6 μl of 50 mM Tris(2-carboxyethyl)phosphine in diH 2 O.

Techniques: Polymerase Chain Reaction, Western Blot, Transgenic Assay, Control, Injection, Muscles, Fluorescence, Labeling, Microscopy