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GndCl-based lysis and in solution digestion

GF Giulia Franciosa
JS Jos G. A. Smits
SM Sonia Minuzzo
AM Ana Martinez-Val
SI Stefano Indraccolo
JO Jesper V. Olsen
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Cell pellet was resuspended in boiling lysis buffer (6 M guanidine hydrochloride, GndCl, Sigma-Aldrich), supplemented with 5 mM tris(2-carboxyethyl)phosphine (TCEP), 10 mM chloroacetamide (CAA), 100 mM Tris-HCl (pH 8.5). Cells were boiled for an additional 10 min and further lysed by micro tip sonication (Vibra-Cell VCX130, Sonics, Newtown, CT) for 2 min with pulses of 1 second on and 1 s off at 80% amplitude. Protein concentration was estimated by Bradford assay (Bio-Rad).

The lysates were digested with Lys-C (FUJIFILM Wako Pure Chemical Corporation) in an enzyme/protein ratio of 1:200 (w/w) for 1 h followed by a 6-fold dilution with 25 mM Tris, pH 8.5, to 1 M GndCl and further digested overnight with trypsin (Sigma-Aldrich) 1:100 (w/w) at 37 °C.

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