Supplementary Figure 3: Optimization of SORT-M using an AlkK tagged E. coli protein extract. | Nature Biotechnology

Supplementary Figure 3: Optimization of SORT-M using an AlkK tagged E. coli protein extract.

From: Labeling and identifying cell-specific proteomes in the mouse brain

Supplementary Figure 3

A. SORT-M for the comparison of click ligands (THPA vs TBTA), reducing agents (TCEP vs sodium ascorbate) and dye concentrations. Reaction conditions; Alexa-647 azide (variable), CuSO4 (1mM), reducing agent (1 mM), ligand (0.1 mM), RT, 2 hrs. B. SORT-M for the optimization of copper concentration and ligand to copper ratio for both TBTA and THPTA ligands. Reaction conditions; Alexa-647 azide (0.1 mM), CuSO4 (varible), sodium ascorbate (1 mM), ligand (0.1 mM), RT, 2 hrs. C. Time course of SORT-M labeling using optimized conditions. Reaction conditions; Alexa-647 azide (0.1 mM), CuSO4 (1 mM), sodium ascorbate (1 mM), ligand (1 mM), RT. D. SORT-M to monitor the effect of aminoguanidine supplement on optimized click conditions. Reaction conditions; Alexa-647 azide. (0.1 mM), CuSO4 (1 mM), sodium ascorbate (1 mM), ligand (1 mM), aminoguanidine (0 or 5mM), RT, overnight. The experiments in AD were performed once with this gel configuration. The optimized conditions were tested ten times.

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