Isoelectric Point Determination

Molecular Mass Determination of Protein Monomer SDS-PACE 

Electrophoresis causes the migration of charged molecules in solution along the %-axis in response to an electric field. The migration rate or velocity v can be obtained from a balance of electrical force and Stokes drag and expressed by Eq. (9.15). 

electrophoresis commonly measures size/charge ratios of migrating molecules (or, better, charge/size ratios (z/dft). The Stokes diameter of a protein [nm] is related to the molar mass Mr [g moh1] by Eq. (9.17). 

The logarithm of the molecular weight of an SDS-denatured polypeptide scales linearly with the polypeptide s Rvalue [Eq. (9.18)]. The R value is calculated as the ratio of the distance migrated by the molecule to that migrated by a low-molecular-weight marker dye-front. 

Separated proteins can be compared to markers, a mixture of proteins with known molecular weights, in adjacent lanes in the same gel. A simple procedure of deter- 

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The isoelectric point, determined by Sumner and Hand (50) to have a value of 5.0-5.1, has been redetermined by the electrofocusing technique (43). The value obtained was 4.8. The solubility is extremely small at this pH, but the urease can be located by its enzymic action. It is a point of interest that the solubility of the isoelectric species increases spectacularly if the sulfhydryl groups are substituted with A-ethylmaleimide (NEM) (43). 

Schmitt, R, Poiger, T., Simon, R., Garrison, A. W., Freitag, D., and Kettrup, A. (1997b). Simultaneous ionization constants and isoelectric points determination of 12 hydroxy-s-triazines by capillary zone electrophoresis (CZE) and capillary electrophoresis isoelectric focusing (CIEF). Anal. Chem. 69,2559-2566. 

Capillary isoelectric focusing (CIEE), Isoelectric point (p7) Proteins and peptides, glycoproteins, monoclonal antibodies, isoelectric point determination, peptide mapping... 

The titration curves are automatically calculated for each 0.1 pH or 0.1 paH unit with a Tektronix TK 31 calculator and two DM 501 multimeters placed in line with the titration assembly. b. Isoelectric Point Determination. Isoelectric points can be meas-... 

Some reported values of the isoelectric point are based on the change by carbon of the pH of buffered solutions, the buffer solution whose hydrogen ion concentration is unaffected being considered to represent the isoelectric pH of the carbon. Values thus obtained range from pH 5 to 9 and appear to depend on the selective adsorb-ability of the separate constituents of the buffer. Therefore, they bear no relation to isoelectric points determined electrokinetically, and have no real significance for such purpose (see 9 16). 

From an applications view point, it should be noted that the membranes are negative over most of the relevant pH range. Similar results were obtained by Kim et al. (1994), who measured the zeta potential through the pores rather than along the surface. However, the isoelectric points determined by those authors were 3.7 and 4.4 to 5.3 for the GVWP and GVHP membranes, respectively. 

FIG. 6 Effect of atmospheric exposure on the isoelectric point (determined by electrophoresis) and point of zero charge (determined by mass titration) for an activated carbon treated in H2 at 950°C. 

CIEF Isoelectric point (pi) Proteins, peptides Glycoproteins MABs Isoelectric point determination Peptide mapping... 

The study of pH effect has been accomplished by some investigators. Some of them find a linear variation with pH, while others reported a plateau at some pH values [30]. Therefore we studied zeta potential of polymers at constant pH, only for isoelectric point determination we provided pH dependence. Also the effect of temperature should not be neglected, e.g. the -potential of silica increases approximately 1.75% per 1°C [30]. It is therefore important to preserve the constant temperature during zeta potential determination. 

Round robin - Electrophoresis and dH measurements - The round robin electrophoresis on the goethite standards prepared at Wisconsin indicated several interesting features. The isoelectric points determined at each of the participants were similar, but measured pH values displayed considerable scatter. The suspected influence of dissolved CO and adducts and methods to overcome the effect of these dissolved species are currently under investigation at Penn State. 

Comparatively few attempts have so far been made to utilize zone electrophoresis for mobility and isoelectric point determinations. It appears that such measurements are possible but not with the same degree of accuracy as in free electrophoresis. The effect of adsorption already discussed above (p. 471) is particularly serious. Furthermore a medium such as filter paper may not have a sufficiently uniform cross-section area. A number of other factors that influence the zone movement may, however, be accounted for by suitable corrections. 

It should be noted that the equilibrium conditions, Eqs. (2) and (6b), have the same form whether there is adsorbed surface charge or not. The magnitudes of (0) and Vc(0), however, will differ in these two cases because of the difference in the charge neutrality conditions, Eqs. (4) and (6a). An important consequence of the above consideration is that in the presence of adsorbed surface charge and constant x potential the space charge potentials Vs(0) and Fc(0) will not be zero at Cf/O), and in fact will not be zero at the same value of silver ion concentration in solution. The definition of isoelectric point therefore becomes ambiguous and it is necessary to define isoelectric point separately for each side of the interface corresponding to values of silver ion concentration in solution where F (0) = 0 or Fc(0) = 0. If only K (0) is measured, as in the electrophoresis experiment, the isoelectric point determined for F (0) = 0 does not ensure that Kc(0) is zero at the same time. 

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