Supplementary Figure 12: Optimizing AlkK based SORT-E in E. coli.
From: Labeling and identifying cell-specific proteomes in the mouse brain
A. Optimizing ADB conjugation to a SORT tagged proteome. Comparison of click ligands (THPA vs TBTA) and ADB concentration. ADB ligation conditions; ADB (variable), CuSO4 (1mM), sodium ascorbate (1 mM), ligand (1 mM), aminoguanidine (5mM), RT, overnight. The ADB was detected by Steptavidin-HRP . This experiment was performed once in this gel configuration. B. SORT-E of E. coli protein extract tagged with AlkK using optimized reaction conditions. ADB ligation conditions; ADB (0.1 mM), CuSO4 (1 mM), sodium ascorbate (1 mM), TBTA (1 mM), aminoguanidine (5mM), RT, overnight. Approximately 30 ug of protein is isolated from an input of 35 mg of E. coli protein. This experiment was performed once in this gel configuration. C. Copious washes of proteins immobilized onto streptavidin beads during SORT-E does not effect the yield of eluted proteins. ADB ligation conditions; ADB (0.1 mM), CuSO4 (1 mM), sodium ascorbate (1 mM), TBTA (1 mM), aminoguanidine (5mM), RT, overnight. Beads washed with 1% SDS in PBS, proteins eluted with 1% SDS in PBS, 50 mM Na2S2O4. *Gel analysis of sampled from the last wash of X number of washes (X = 3, 6, 9, 12 or 15). Proteins were visualized by SyproRuby on SDS-PAGE. The experiment was performed twice in this configuration.
