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To remove the PEG8000 prior to nucleic acid extraction a buffer exchange was attempted by the addition of 4 ml of SM-plus to the sample and centrifugation in a Amicon® Ultra Centrifugal Filter (50,000 MWCO).
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Buffer exchange was repeated once.
The protein solution after buffer exchange was subjected to the peptide-cleavage process.
The eluted protein was concentrated, and a buffer exchange was performed with 25 mM phosphate buffer containing 10 µM FAD.
Imaging was initiated as soon as the buffer exchange was complete.
A quantitative analysis of the exchange kinetics was attempted but led to insufficient fitting results because the fast exchange component could not be described appropriately.
The exchanged buffer exchanged was 0.01 × PBS/0.01 mM DTT/2.5 mM imidazole/0.1% glycerol.
Other buffers having the same pH value such as Britton Robinson buffer and phosphate buffer were attempted and compared with 0.2 M borate buffer.
In patients, who were hemodynamically stable, prone positioning was attempted to optimize gas exchange.
This stylet was then exchanged with a locking stylet and lead extraction was attempted.
Exchanges between configurations were attempted every 200 steps.
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