Ancillary Agreement For Plasmids Containing Fp Materials

These plasmids were created by your colleagues. Please confirm the Principal Investigator, quote the article describing the plasmids and add additives to the documents and methods of your future publications. 1) The Regents of the University of California own certain GFP and RFP (FP MATERIAL) materials through their San Diego campus (UCSD) and reserve ownership rights to FP material contained in all MATERIALs derived from THE RECIPIENT. FP material is covered by certain patents granted and pending patents belonging to UCSD and other third parties. For the cleaning of the CLC-7/Ostm1 complex proteins, 2 litres of transfinted HEK293F cells were collected by centrifugation at 3000g. The cell pellets were placed in lyse pads containing 20 mM hepes (pH 7.4) and 150 mM of NaCl, Leupeptine (1 g/ml), pepstatin (1.5 g/ml), aprotinin (0.84 g/ml), 0.3 mM phenyl-mulfonylfluopreure and rehydrated by sound at 5 min. The cell membrane was shoveled for 1 hour after an ultra-trifugation of 100,000g. The membrane was used in tampons of 20 m hepes (pH 7.4), 150 mM of NaCl, 2 mM of dithiothréitol (DTT) and 1% (w/v) of digitonine for 2 hours with a gentle rotation at 4oC. After an ultracentrifugation at 100,000g for 20 minutes, overhang with strep-tactin sepharose (IBA) was incubated for 1 hour with a gentle rotation at 4oC. The resin was washed with a washable tampon of 20 mM hepes (pH 7.4), 150 mM naCl, 2 mM DTT and 0.1% (w/v) of digitonine.

The CLC-7/Ostm1 target complex was etalized with a washing pad plus 5 mM d-desthiobiotin (IBA) and concentrated on a final volume of about 100 l. The final protein was applied to size elimination chromatography (Superpose-6 10/300 GL, GE Healthcare) in tampons containing 20 mM hepes (pH 7.4), 150 mM of NaCl and 0.1% of digitonine. The peak for the CLC-7/Ostm1 complex was collected for further cryo-microscopy analyses. To facilitate verification of CLC-7 and Ostm1 interactions, we are producing a CLC-7 plasmid that contains a C-terminal Strep beacon and an Ostm1 plasmid with a c-Terminal Flag tag. Point mutations were introduced by polymerrase chain reaction, and the resulting constructions were CLC-7 (E416A) and Ostm1 (Y300A). In short, we will transcribe 50 ml HEK293F cells with the following combinations: WT CLC-7 and WT Ostm1, WT CLC-7 and Y300A (Ostm1), E416A (CLC-7) and WT Ostm1. The transfied cells were lysed with 1% digitonine in 1 ml lysis buffers [25 mM tris (pH 7.8), 150 mM NaCl, 1 mM EGTA, 1 mM EGTA, cOmplete proteemia inhibitors]. The cell lysat was incubated on the ice for 30 minutes and centrifugal at 4 to 20,000 g.

Overhang was incubated for 2 hours at 4oC with the anti-Strep magnetic agarose (Thermo Fisher Scientific). The beads were picked up on the magnet, washed three times with 1 ml of lysis pad with 0.1% digitonine and 150 SDS gel load pad for Western Blot. The same process was performed in the pull-down test with the anti-flag magnetic beads (Thermo Fisher Scientific) transmitted in vitro. For Western Blot`s analysis, the proteins were subjected to an electrophoresis gel of 4 to 20% polyacrylamide-gel SDS (GenScript) and transmitted to a polyvinylide difluoline membrane (millipore).

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