Could insulin come in a pill? How a molecule that mimics insulin may advance diabetesresearch WEHI_research NatureComms
followed by the sequence SSDYKD was synthesized and then cloned into the vector pJ201 . Both genes were then individually transferred into the BamH1/Xba1 sites of the plasmid pNCM02 for independent transformation intocells were then screened for over-expression of the expected domain by Western blot using antibodies 9E10 and KM5-1C7-8-5 at a concentration of 0.
. The highest-expressing colonies were then stored as glycerol stocks. For 1 L scale-up, each glycerol stock was used to inoculate 2 mL of 2SY broth containing 10 μg mLneomycin sulfate , followed by incubation overnight at 30 °C at 120 rpm on a rotating platform. 0.2 mL of these respective cultures were then used to inoculate a further 20 mL of 2SYnm broth and, once sufficiently grown, 5 mL of this inoculum was used to inoculate a further 500 mL of 2SYnm broth in Tunair™ flasks .
and the desired Fv 83-7 eluted with c-myc peptide prepared in Tris-buffered saline plus azide . Fractions were combined with one tablet of cOmplete protease inhibitor cocktail . Fractions from Superdex 200 10/300 SEC examined by SDS-PAGE showed the presence of two bands of molecular weight 14 and 16 kDa, respectively, indicating a correctly-formed Fv eluting at 22 kDa. The c-myc tag was then removed from the 83-7 VH domain of the Fv 83-7 as follows. The Fv 83-7 was diluted to ~ 4 mg mLand 0.
4-μL volumes of the respective samples were applied to UltrAuFoil R1.2/1.3 300-mesh grids that were prior glow discharged in a Pelco easiGlow device at 10 mA for 90 s or at 15 mA for 30 s . Grids were blotted for 3 s at −3 blot-force setting in a Vitrobot mark IV before being plunge frozen in liquid ethane.CryoEM imaging was performed using a Titan Krios equipped with a Gatan K2 Summit™ camera, a Quantum-GIF energy filter and the EPU 2 data acquisition software.
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