To assess the stability of serum samples stored at 23°C, 4°C, —20°C, and —80°C and liquid nitrogen (LN2), samples were simultaneously thawed and separated using precast NuPAGE™ (Invitrogen) SDS gels. For low molecular weight protein separation, 4 mg serum was separated using 10% Bis-Tris NuPAGE™ gels, electro-phoresed using MOPS running buffer, and stained with SYPRO Ruby fluorescent gel stain (Molecular Probes). For analysis oflarge proteins, 1 mg of serum was run on 3-8% Tris-Acetate NuPage™ gradient gels with Tris-Acetate running buffer, and silver stained (Silver Quest silver staining kit; Invitrogen). SYPRO Ruby stained gels were scanned using the ProXpress Proteomic Imaging System (Perkin Elmer Life and Analytical Sciences).
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