BioMed Research International / 2012 / Article / Fig 4

Review Article

Proteomics Shows New Faces for the Old Penicillin Producer Penicillium chrysogenum

Figure 4

Optimization of the protein extraction protocol from mycelia of P. chrysogenum. (a) Extraction and purification of protein samples with ‘‘Clean-Up Kit” (GE Healthcare), based on Kniemeyer and coworkers [66] and silver stained. As sample buffer was used, 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 0.8% (v/v) IPG buffer pH 3–10 nonlinear (NL) (GE Healthcare), 40 mM Tris, 1 mM EDTA, and 20 mM DTT [75]. (b) The extraction is based on the method described by Fernández-Acero and coworkers [63] by using mortar gridding and phosphate buffer, plus blue silver Coomassie colloidal staining [76]. As sample buffer was used, 8 M urea, 2% (w/v) CHAPS, 0.5% (v/v) IPG buffer pH 3–10 NL (GE Healthcare), 20 mM DTT, and 0.002% bromophenol blue [69]. Precision plus protein standards (Bio-Rad) were used as markers. The molecular mass is indicated in kilo-Daltons (kDa). Note the problematic regions observed with the extraction method A, which are highlighted by arrowed square brackets (left and bottom), Barreiro et al., 2011.

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