When optical tweezers were used, the ATP buffer was supplemented with NeutrAvidin-functionalized silica microspheres.
Wild-type and mutant FICD/HYPE were expressed and purified as EfFIC, except that the lysis buffer was supplemented with 1 mM dithiothreitol (DTT) and 0.02 % Triton X-100 and the other buffers were supplemented with 1 mM DTT.
For purification of the hydantoinase, the buffer was supplemented with 1 mM ZnSO4, while for the carbamoylase 5 mM DTT were added.
Lysis buffer was supplemented with Complete EDTA-free protease inhibitor cocktail (Roche).
The lysis buffer was supplemented with the Complete Protease Inhibitor Cocktail, EDTA-free (Roche).
For immunoprecipitation assays, this lysis buffer was supplemented with 10% glycerol.
To analyze co-immunoprecipitation of CSP and VAMP2, the lysis buffer was supplemented with 1% SDS.
Lysis buffer was supplemented with protease and phosphatase inhibitors, including 1 mM sodium fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 mM PMSF (Phenylmethanesulfonyl fluoride).
To solubilize Arabidopsis membrane proteins, the membrane resuspension buffer was supplemented with 0.5% (w/v) 1-Palmitoyl-2-hydroy-sn-Glycero-3-Phosphocholine (LPC) (Avanti Polar Lipids, Inc) or 0.6% (w/v) Octyl β-D-glucopyranoside (OG) (Sigma).
Prior to the second-dimension electrophoresis, strips were equilibrated twice in 2 ml equilibration buffer [20% (v/v) glycerol, 2% (w/v) SDS, 6 M urea, 0.375 M Tris/HCl, pH 8.8] for 10 min. This equilibration buffer was supplemented with 130 mM DTT for the first equilibration and with 135 mM iodoacetamide for the second one.
Because lysozyme has a higher than average percentage of formaldehyde-reactive residues than FFPE tissue or the tissue surrogate mixture, the lysozyme tissue surrogates were heated at 100°C for 2 h and the 50 mM Tris-HCl, pH 4, 2% SDS extraction buffer was supplemented with 0.2 M glycine as an additional formaldehyde scavenger.
