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Buffer exchange was repeated once.
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This exchange is repeated several times.
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.
To obtain an estimate for the standard errors, each exchange experiment was repeated twice.
The exchanged buffer exchanged was 0.01 × PBS/0.01 mM DTT/2.5 mM imidazole/0.1% glycerol.
This step was repeated three times to ensure complete buffer exchange.
The processes was repeated twice more, once with wash buffer, then once with exchange buffer (20 mM sodium phosphate at pH 7.5 with a quantity of detergent expected to yield 0.4 mM concentration of micelles (Tables 1 and 2)).
This step was repeated using the same buffer with 4% iodoacetamide in place of 1% DTT.
After each force-extension curve was obtained, the buffer in the flow cell was exchanged to increase the concentration of salt, and the experiment was repeated.
The procedure was repeated with reaction i and j exchanged.
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