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In cases where larger higher amounts of cells were recovered, extracts were also analyzed for receptor proteins.
The recovered extracts were pooled, dried by vacuum centrifuge, resuspended in 10 μL of 0.1% formic acid, and analyzed by mass spectrometry.
The recovered extracts were then reduced in a rotary evaporator and finally stored in airtight containers at 4°C for further use.
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Seventeen extracts were not evaluated for their cytotoxic activity because of the yield of the recovered extract (less than 1 mg).
The recovered extract was evaporated under a stream of nitrogen (Turbovap) and the gravimetric extract weight was recorded after further drying over night in a vacuum oven at 40 °C.
Additional file 6: Mass of extracts recovered upon extraction of PdGAUT12.1 -KD AIR.
The recovered crude extracts were kept at −80 °C until use.
Protein concentration in recovered protein extracts was determined using BioRad Protein Assay Dye Reagent Concentrate Bio-Rad Laboratoriess) using Bovine Serum Albumin (BSA) as a standard.
As shown in Figure 1E, RPC1 was specifically recovered from extracts supplemented with Maf1 (compare lanes 2 and 3).
The recovered ethanol extracts were evaporated under reduced pressure to yield 25.3 g.
Furthermore, full length APOBEC3G is efficiently recovered from extracts by the extended linear polyU RNA, a reflection of its ability to oligomerise in an RNA dependent fashion, whereas AID recovery is not enhanced by its binding to linear polyU RNA.
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