Size Estimation of Native Proteins and Enzymes

The size of native proteins can be deduced from their migration behaviour in homogeneous or gradient gels. Both methods have the advantage that crude tissue or cell extracts can be used as the protein source, provided a specific staining method exists with which they can be located in the gel after elec-trophoresis. The method with homogeneous gels uses a number of gels of different PA concentration in the range of 4-35% T and estimates the relative elec-trophoretic mobility referred to Bromophenol blue (RF value) of a set of marker proteins and the sample protein(s). From these values the gel concentration is estimated at which the electrophoretic mobility is zero (or would become zero). This is achieved by plotting the logarithm of the % T concentration (log T) in which the mobility is measured against the respective RF value. In the underlying linear function (log T = -kxRF + log Tlim), the value of Tlim represents the exclusion limit, the %T concentration at which protein mobility stops. The Tlim values cal culated for a number of marker proteins can be correlated to their corresponding Stokes radii (RS) to obtain a calibration line. A linear function is obtained when RS is plotted against the reciprocal of Tlim (RS = ax 1/Tlim + b). Into this equation the exclusion limit of a sample protein is inserted and this then allows calculation of the corresponding Stokes radius.

Polyacrylamide gradient gel electrophoresis can also be used to estimate the molecular size of nondenatured proteins, provided it is performed in a time-dependent way. The following physicochemical properties of native proteins (enzymes) are obtainable:

2. hydrodynamic radius (Stokes radius (RS));

3. frictional coefficient (f/fo) (molecular eccentricity, considering the molecular shape as a rotational ellipsoid and f/fo as the quotient of the ratio of the two half axes of the rotational ellipsoid, f = half axis of ellipsoid, fo = half axis of circle);

4. isomeric nature of multiple protein forms (size isomers or charge isomers);

5. free electrophoretic mobility (and nett negative charge (valence Z, charge Q)) at the pH value of the electrophoresis.

The mathematical procedures used to calculate these parameters are bound by several preconditions:

1. The PA gradient increases linearly (at a constant ratio of acrylamide to Bis). The gradient range however, can be chosen freely.

2. The electrophoretic pH value and the voltage gradient are chosen in a way that marker and sample proteins migrate sufficiently.

3. The same buffer system has been used as gel and electrode buffer, if net charges are to be obtained.

4. The sizes of the marker and sample proteins fit the pore range of the PA gradient.

5. Marker and sample proteins have migrated on the same gel slab.

6. Parts of the gel slab which have been cut into two or more parts and stained differently are re-equilibrated to the original gel length before protein migrations are measured.

7. Approximately 10 (or more) time-dependent migration distances of marker and sample proteins are accurately measured.

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Solar Panel Basics

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