Samples were prepared for MS as follows. After the final wash, the beads were resuspended in 100 µl of 50 mM ammonium bicarbonate (Thermo Fisher Scientific), and the proteins on the beads were reduced with 10 mM DTT for 30 min at room temperature and alkylated with 55 mM iodoacetamide (Sigma-Aldrich) for 30 min in the dark. Protein digestion was performed with sequencing grade modified trypsin (Promega) at 1/50 protease/protein (wt/wt) at 37°C overnight. After trypsin digestion, the beads were washed twice with 100 µl of 80% acetonitrile (Thermo Fisher Scientific) in 1% formic acid (Thermo Fisher Scientific), and the supernatants were collected. Samples were dried in Speed-Vac (Thermo Fisher Scientific) and desalted and concentrated on a C18 Tip (Thermo Fisher Scientific).
50 mm ammonium bicarbonate
Manufactured by Thermo Fisher Scientific
50 mM ammonium bicarbonate is a laboratory reagent. It is an aqueous solution of ammonium bicarbonate with a concentration of 50 millimolar.
Automatically generated - may contain errors
Lab products found in correlation
Iodoacetamide, by Merck Group (2 mentions)
Speedvac, by Thermo Fisher Scientific (2 mentions)
Sequencing grade modified trypsin, by Promega (2 mentions)
Formic acid, by Thermo Fisher Scientific (1 mentions)
Acetonitrile, by Thermo Fisher Scientific (1 mentions)
C18 tip, by Thermo Fisher Scientific (1 mentions)
Pierce c18 tip, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
2 protocols using 50 mm ammonium bicarbonate
1
Protein Purification and Trypsin Digestion
2
Protein Preparation for Mass Spectrometry
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Samples were prepared for MS as previously described (Kendrick et al., 2019 (link)). After the final wash the beads were resuspended in 100 μl of 50 mM ammonium bicarbonate (Thermo Fisher Scientific) and the proteins on the beads were reduced with 10 mM DTT for 30 min at room temperature and alkylated with 55 mM iodoacetamide (Sigma) for 30 min in the dark. Protein digestion was carried out with sequencing grade modified trypsin (Promega) at 1/50 protease/protein (wt/wt) at 37°C overnight. After trypsin digestion, the beads were washed twice with 100 μl of 80% acetonitrile (Thermo Fisher Scientific) in 1% formic acid (Thermo Fisher Scientific) and the supernatants were collected. Samples were dried in Speed-Vac (Thermo Fisher Scientific) and desalted and concentrated on Thermo Fisher Scientific Pierce C18 Tip.
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