*4.10. Streptavidin Enrichment*

Streptavidin enrichment was by method adopted from the method described by Speers and Cravatt [35]. Probed protein precipitates were dissolved in PBS using 600 μL of 0.2% SDS. Then 100 μL of streptavidin agarose resin (Pierce™ 20347, Thermo Fisher Scientific, Waltham, MA, USA) was added and mixed for 1 h in a shaker at RT. It is followed by washing with 1 mL of 1× PBS with centrifugation (for 1 min at 2500× *g*) in between and each time collection of supernatants. Finally, the streptavidin beads were eluted by adding 100 μL of 2× SDS-loading buffer, boiling in a water bath for 10 min. Cooling at 4 ◦C and centrifuging at 2500× *g* for 1 min. The supernatants were collected and stored at −20 ◦C until use.

#### *4.11. SDS-PAGE and Staining*

Protein samples collected from the previous step were resolved in 10% SDS-PAGE gel and visualized by boiling in 0.25% Coomassie Brilliant Blue (CBB) stain for 5 min [26]. The blue-stained gel was washed twice by boiling it with distilled water for 5–10 min. Protein bands from sample columns were cut and stored (at −20 ◦C) until the identification experiment.
