Estimation of Stokes Radius and Molecular Mass

The maximum migration distance of globular proteins is related to the maximum gel pore radius at the respective gel concentration (cf. Figure 1). Therefore, the maximum migration distances (Dmax) of proteins can be correlated to their Stokes radius (RS). A linear relationship is obtained if the logarithm of the maximum migration distance (ln Dmax) of proteins is plotted versus the logarithm of their Stokes radius (ln RS):

Figure 10 (A) Plot of migration distances (D (mm)) of marker proteins and (B) of five different carbonic anhydrases versus times of electrophoresis (t (h)) in a linear PA gradient gel of 4-30% T. Conditions of electrophoresis are given in Figure 9. Migration distances and times of electrophoresis as listed in Table 2. OVA, Ovalbumin; BSA, bovine serum albumin; LDH, lactate dehydrogenase; CAT, catalase; FER, ferritin; TYR, thyroglobulin. Marker proteins and carbonic anhydrases were migrated on the same gradient gel. Purified enzyme preparations (Sigma, Munich, Germany) comprised carbonic anhydrases from bovine (I-III), rabbit (III, IV), human (V) and canine (V) erythrocytes. (Reproduced with permission from Chrambach etal. Advances in Electrophoresis Vol 4: pp 351-358.)

Figure 10 (A) Plot of migration distances (D (mm)) of marker proteins and (B) of five different carbonic anhydrases versus times of electrophoresis (t (h)) in a linear PA gradient gel of 4-30% T. Conditions of electrophoresis are given in Figure 9. Migration distances and times of electrophoresis as listed in Table 2. OVA, Ovalbumin; BSA, bovine serum albumin; LDH, lactate dehydrogenase; CAT, catalase; FER, ferritin; TYR, thyroglobulin. Marker proteins and carbonic anhydrases were migrated on the same gradient gel. Purified enzyme preparations (Sigma, Munich, Germany) comprised carbonic anhydrases from bovine (I-III), rabbit (III, IV), human (V) and canine (V) erythrocytes. (Reproduced with permission from Chrambach etal. Advances in Electrophoresis Vol 4: pp 351-358.)

where ln Dmax equals eb of eqn [7], and m and b represent the slope and intercept of the straight line (Figure 12).

It has been shown that a similar equation correlates the logarithm of the maximum migration distance (ln Dmax) to the logarithm of the molecular mass (ln Mr):

where ln Dmax equals eb of eqn [7], and z and c represent the slope and intercept of the straight line (Figure 12).

Knowing the maximum migration distance of any native globular protein, the calibration line can be used to calculate the molecular mass of the protein by inserting the calculated ln Dmax value and the values of the slope (z) and the intercept (c) of the calibration line into the equation ln Dmax =— z x ln Mr + c (Table 3) or inserting the ln Dmax value and the values of the slope (m) and the intercept (b) of the calibration line into the equation ln Dmax = —mx ln Rs + b (Table 4).

When using PA gradients of 4-30% T and a buffer of pH 8.4 (45 mmol Tris, 40mmolL~1 boric acid, 1.25 mmol L"1 EDTA-Na2, pH 8.4) a number of markers can be used, ranging from carbonic an-hydrase (Mr 30 000, Rs 3.05) to thyroglobulin (Mr 669 000, Rs 8.50; Table 5). ^-Galactosidase (Mr 116000, Rs 4.23) and carbonic anhydratase (Sigma, St Louis, MO, USA) are run in the same lane and the other marker proteins are run in a separate one. The marker proteins bovine serum albumin, lactate de-hydrogenase, catalase, ferritin and thyroglobulin can be obtained as a freeze-dried mixture (Amersham Pharmacia Biotech, Freiburg, Germany) and dissolved in a solution of pure ovalbumin (Boehringer, Mannheim, Germany). Separation times depend on the voltage gradient and may range from 0.5 to more than 20 h (Table 2).

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