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Fig. 7 | Clinical Proteomics

Fig. 7

From: Freeze-dried plasma proteins are stable at room temperature for at least 1 year

Fig. 7

Western blot against the mature C4B protein chain in human EDTA plasma over the time course of degradation at room temperature. The monoclonal antibody against C4B was biotinylated and detected with Streptavidin-HRP. The arrow indicates the expected C4B peptide after proteolysis. Lanes: MW, Molecular weight marker as indicated; A, Streptavidin HRP alone; 1, time 0; 2, 1 h; 3, 1 h; 4, 4 h; 5, 8 h; 6, 24 h; 7, 36 h; 8, 48 h; 9, 72, hr; 10, 96 h. Molecular weight markers for a 9% Tricine SDS-PAGE gel are shown in kilodaltons (kDa). The Western blot analysis showed the increased formation of mature C4B with time and the development of an additional band with time at room temperature confirming the proteolytic processing of C4B. Complement C4 is expressed as a high molecular mass preproprotein that reacts poorly with the antibody raised against the mature protein that exits in blood as a 79 kDa mature chain in close agreement with the most intense band in the Western blot that appears roughly in line with the 75 kDa molecular mass marker. Proteolytic cleavage of the C-terminal domain from C4B starting at NGFKSHALQLNNR to the carboxyl terminus of the C4B protein should yield a 63 kDa protein (see arrow). Control blots stained with Coomassie Blue confirmed equal loading of all lanes (see Additional file 1: Fig. S1). These data confirm the finding shown in Table 2 and Fig. 2. The manufacturer (THERMO) indicates the antibody recognizes an 88, 75 and 33 kDa form of C4B in good agreement

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