Fig. 4: Light-controlled affinity chromatography (LCAC) to purify SH2-tagged proteins using cobalt-immobilized OptoMB.
From: Development of light-responsive protein binding in the monobody non-immunoglobulin scaffold
a Schematic diagram of functionalization of LCAC Co2+-agarose beads using His6-OptoMBV416I_G528A_N538E (OptoMBTriple) containing a SUMO tag (S) and LCAC procedure involving dark incubation of crude E. coli extract containing YFP-SH2, washing in the dark, and eluting with blue light in batch or column (Supplementary Fig. 4). SDS-PAGE was used to resolve the fractions from each purification step. b, c SDS-PAGE gel of LCAC-purified YFP-SH2 (b) and SH2-Pdc1p (c). Molecular weight markers (M), lysate (L), unbound flow through (FT), washing step in the dark (Dark Wash), two consecutives light elution aliquots (Light Elution 1 and 2), and the imidazole elution (I) were resolved in 12% SDS-PAGE. Purifications were repeated at least three times observing similar results.
