9), 10mM KC1, 2mM MgCl2, 1 mM dithiothreitol
5,1 mM EDTA, 1 M NaCl, 20% glycerol) containing 2 mM P-mercaptoethanol, and then against buffer B containing 1 mM dithiothreitol
Protein bands in the appropriate relative molecular weight range were manually excised, reduced with 5 mM dithiothreitol
(DTT) and alkylated with 50 mM iodoacetamide (Bio-Rad Laboratories), digested with trypsin (Promega Corp.
A20-[micro]L aliquot was diluted to 300 [micro]L with 50 mmol/L ammonium bicarbonate (pH 8), reduced with 10 mmol/L dithiothreitol
(DTT) at 60[degrees]C for 45 min, alkylated with 10 mmol/L iodoacetamide at room temperature for 30 min in the dark, reduced again with 5 mmol/L DTT at room temperature for 30 min in the dark, and digested overnight at 37[degrees]C with modified trypsin at a 1:50 (wt/wt) trypsin:total protein ratio.
2) and lysed in a lysis buffer (8 M urea, 4% CHAPS, 40 mM dithiothreitol
0, 1 mM phenylmethylsulfonyl fluoride (PMSF) (Roche, Germany), 50 x Protease inhibitor (Roche, Germany), 200 mM dithiothreitol
(DTT) (GE Healthcare, Sweden).
6 [micro]L of 1 tool dithiothreitol
at 65[degrees]C for 15 min, neutralized with 8.
(DTT), propyl thiouracil (PTU) were purchased from Sigma Chemical Co.
5 mM dithiothreitol
, 100 [micro]g/mL phenylmethylsulfonyl fluoride, and 2 [micro]g/mL leupeptin.
3]), trifluoroethanol (TFE), iodoacetamide (IAA), trifluoroacetic acid (TFA), and TPCK-treated trypsin (T-1426) from Sigma-Aldrich; dithiothreitol
(DTT) and formic acid from Fluka; fetal calf serum (FCS) from Invitrogen (Gibco 10437-028); Zwittergent detergent 3-16 (Z 3-16) from Calbiochem; and highly purified A1AT reference material from Athens Research & Technology (16-16-011609).
4], 1 mM dithiothreitol
, the desired concentration of extract, and an NADPH-generating system composed of 0.
The beads were resuspended in 100 [micro]L of 10 mmol/L dithiothreitol
and incubated at 56 [degrees]C for 45 min.