Click Chemistry Protocol for Azide-labeled Samples

NHS-Azide-injected samples were processed for click chemistry according to the following protocol, modified from previous studies (Dieterich et al., 2010 (link); Hinz et al., 2012 (link)). Sections or wholemounts were transferred to Eppendorf tubes or six-well plates with reaction mixture composed of 100 μm tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA, Sigma) dissolved in 4:1 tBuOH/DMSO (Sigma), 100 μm CuSO₄ (Sigma), 1.25 μm Alexa Fluor 488 alkyne (Invitrogen), and 250 μm tris(2-carboxyethyl)phosphine (TCEP, Sigma). The reaction proceeded overnight at room temperature.

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Shah S.H., Schiapparelli L.M., Ma Y., Yokota S., Atkins M., Xia X., Cameron E.G., Huang T., Saturday S., Sun C.B., Knasel C., Blackshaw S., Yates J.R., Cline H.T, & Goldberg J.L. (2022). Quantitative transportomics identifies Kif5a as a major regulator of neurodegeneration. eLife, 11, e68148.

Publication 2022

tBuOH/DMSO CuSO4 Alexa Fluor 488 alkyne

Alexa fluor 488 Alkyne Azide Dmso Methyl amine Phosphine Tbta Tcep Tris

Corresponding Organization :

Other organizations : Vision Eye Institute, Stanford University, University of California, San Diego, Scripps Institution of Oceanography, Johns Hopkins Medicine, Johns Hopkins University

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

None explicitly mentioned

control variables

Sections or wholemounts were transferred to Eppendorf tubes or six-well plates with reaction mixture composed of:
100 μm tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA, Sigma) dissolved in 4:1 tBuOH/DMSO (Sigma)
100 μm CuSO4 (Sigma)
1.25 μm Alexa Fluor 488 alkyne (Invitrogen)
250 μm tris(2-carboxyethyl)phosphine (TCEP, Sigma)

controls

No positive or negative controls were explicitly mentioned in the protocol.

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