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Prior to injection, the sample was buffered with borate at pH 10.2, and primary or secondary amino acids were derivatized with ortho-phthalaldehyde (OPA) or 9-fluorenylmethyl chloroformate (FMOC), respectively.
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In aqueous solutions, such as H2O/D2O, the δH highly depends on pH; therefore, the samples are buffered before analysis.
The sample was buffer exchanged using FASP 1 several times by spinning the tube at 7000 g to remove detergents.
For NMR studies the protein samples were buffered to pH 6.8 with 20 mM sodium phosphate.
The final apo wtAidB sample was buffer exchanged with AidB buffer using a PD-10 column.
The protein samples were buffer exchanged into 0.1 M ammonium acetate (AA) buffer (pH 7.4) using either size-exclusion chromatography spin columns (Bio-Rad) or Amicon Ultra-4 centrifugal filter units (EMD Millipore).
Finally, the sample was washed with TE buffer in the same way.
The sample was then diluted in RLT buffer and ethanol.
The sample was diluted (1 1) with the sample buffer.
Then, the sample was resuspended in SDS-Laemmli sample buffer.
The sample was mixed with non-reducing sample buffer and boiled immediately at 95°C.
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