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. Author manuscript; available in PMC: 2007 Nov 1.
Published in final edited form as: Biochim Biophys Acta. 2006 Sep 16;1764(11):1741–1749. doi: 10.1016/j.bbapap.2006.09.005

Figure 1.

Figure 1

Size-sieving chromatography of recombinant bovine factor B on the Sephacryl S-200 HR column (1.5 x 95 cm). A, A major portion of the recombinant bovine factor B sample, obtained after overnight digestion of NusA-factor B fusion with thrombin, eluted at the beginning of the 0-0.3 M NaCl gradient from the ion-exchange chromatography on DEAE-Sepharose Fast Flow column; it was collected, concentrated and reapplied to the size-sieving Sephacryl S-200 HR. The elution profile was monitored by UV at 280 nm. The polypeptide composition of the fractions was analyzed by 15% SDS-PAGE followed by staining with Coomassie brilliant blue. The fractions with recombinant bovine factor B are shown. Inset shows the calibration curve of the column using the molecular weight standards. The molecular weight of factor B eluting in fraction 43 was estimated to be 56 kDa. B, A minor portion of the recombinant bovine factor B sample obtained from the thrombin digest of the NusA-factor B fusion eluted with 0.05-0.1 M NaCl concentration from the ion-exchange chromatography on DEAE-Sepharose Fast Flow column; it was collected, concentrated and reapplied to the size-sieving Sephacryl S-200 HR. The elution profile and the polypeptide composition of the fractions were analyzed as in panel A. Inset shows the calibration curve of the column using the molecular weight standards. The molecular weight of factor B eluting in fraction 47 was estimated to be 34 kDa. The molecular weight standards employed for calibration of the Sephacryl S-200 HR column were as follows: 2,000 kDa, blue dextran, 158 kDa, aldolase, 67 kDa, albumin, 43 kDa, ovalbumin, 25 kDa, chymotrypsinogen A, 13,7 kDa, ribonuclease A.