Universal calibration of size-exclusion chromatography for proteins in guanidinium hydrochloride including the high-molecular-mass proteins titin and nebulin

A Method for the Quantitation of Charge by Size Exclusion Chromatography Demonstrated with Components of Ficoll 400

“…It can be seen from Figure 8, as well as from Figure 4, that the pullulans, ficoll F17-17 and myoglobin all fall within this margin and errors are essentially dominated by flow instability. Our performance is quite comparable to other equipment and investigations [39,65,66] and future technology would have to produce at least a fourfold improvement in order that flow rate instability not be the limiting technique factor. As it is unlikely that the mechanics of pump design could be substantially improved, performance will have to be advanced either with a flow feedback system [67] or simply by replacing the fixed time base in current chromatography with accurate monitoring of actual volume flow.…”

“…In previous studies, the apparent size of proteins was found to vary linearly with I -1/2 under conditions of electrostatic repulsion between solute and gel, finally reaching an apparent size independent of I. [9,32,39] For a variety of proteins, this apparent size closely correlates with Stokes' radii and was observed at I A 0.1-0.2 M, regardless of net protein charge. Similar behavior is observed in the present case inasmuch as the curves for the ionic strength dependence of the pullulan-calibrated R eff values converge upon the known geometric size at about I = 100 mM.…”

“…[38] Stokes radius (R s /nm) = 1.91; quoted by ref. [39] Ovalbumin: net charge Q at pH 8.0, 25 8C (electrophoretic) = -14 l 2; based on measurements by Longsworth [40] a mobility of -0.6 lm N cm N V -1 N sec -1 at pH 8.0, I = 100 mM, 0 8C was used for calculation with Equation 2 (which is not strictly applicable at high ionic strength). The discrepancies claimed in ref.…”

“…[41] pI = 4.7; at 25 8C, [38] the pI increases with increasing ionic strength. Stokes radius (R s /nm) = 2.96; from critically selected sedimentation coefficients [43,44] together with a sequence molecular weight of 44 310 including carbohydrate plus two phosphates groups extra [39] and a partial specific volume of 0.748 mL g -1 .…”

Preparation and characterization of new immunoprotecting membrane coated with amphiphilic multiblock copolymer

“…In the literature, it was reported that the molecular size of IgG is similar to that of 44 kDa dextran. 33 Thus, the extent of rejection of IgG to the membranes can be approximated from the rejection of 44 kDa dextran. Under this assumption, PE20PT20-D membrane as an immunoprotecting membrane would be appropriate because its MWCO for 44 kDa dextran at day 40 is close to the molecular size of IgG.…”