Author: Xiao Huang; Jasper Z. Williams; Ryan Chang; Zhongbo Li; Eric Gai; David M. Patterson; Yu Wei; Wendell A. Lim; Tejal A. Desai
Title: DNA-scaffolded biomaterials enable modular and tunable control of cell-based cancer immunotherapies Document date: 2019_3_23
ID: 5bw7umap_57
Snippet: His-tag GFP were expressed by Escherichia coli BL21 (DE3) (Novagen) transduced with pRSET-EmGFP vector (ThermoFisher, #V35320) in E. coli expression medium (MagicMedia, Invitrogen #K6803), and extracted using cell lysis reagent (Sigma, #B7435) followed by the purification using nickel-nitrilotriacetic acid affinity chromatography (Invitrogen #R90115). The MAL-PEG4-NHS linker (Quanta Biodesign) was mixed with GFP at 30-fold molar excess, and incub.....
Document: His-tag GFP were expressed by Escherichia coli BL21 (DE3) (Novagen) transduced with pRSET-EmGFP vector (ThermoFisher, #V35320) in E. coli expression medium (MagicMedia, Invitrogen #K6803), and extracted using cell lysis reagent (Sigma, #B7435) followed by the purification using nickel-nitrilotriacetic acid affinity chromatography (Invitrogen #R90115). The MAL-PEG4-NHS linker (Quanta Biodesign) was mixed with GFP at 30-fold molar excess, and incubated at 37°C for 1 hour followed by the size-exclusion chromatography to remove the excess. 3'Thiolmodified complementary DNA was decapped using the protocol described above, and reacted with modified GFP at 1:10 molar ratio at 37°C for 1 hour followed by 4°C overnight. The next day, GFP-DNA conjugates are purified using nickelnitrilotriacetic acid affinity chromatography to remove unconjugated DNA. Protein-DNA conjugates were analyzed through SDS-PAGE gel electrophoresis (Genscript #M42012L) with SYPRO Ruby dye staining (ThermoFisher #S21900).
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