Continuous optical scanning in polyacrylamide gel electrophoresis: Estimation of the apparent diffusion coefficient of β-lactoglobulin B
✍ Scribed by Betty Chen; Andreas Chrambach; David Rodbard
- Publisher
- Elsevier Science
- Year
- 1979
- Tongue
- English
- Weight
- 935 KB
- Volume
- 97
- Category
- Article
- ISSN
- 0003-2697
No coin nor oath required. For personal study only.
✦ Synopsis
The continuous scanning apparatus developed by Catsimpoolas was applied to an analysis of the concentration profiles of a protein, ~-la~to~lobulin B, while it was subjected to polyacrylamide gel electrophoresis (PAGE) in a multiphasic buffer system. Continuous optical scanning in PAGE permitted reliable estimation of the standard deviation of the concentration profile (u), the relationship between crZ and time, and the apparent diffusion coefficient, D', derived from u*, as the current density varied from 2 to 9 mA/cm", protein load varied from 250 to 900 &cm'. and the ionic strength varied from 0.015 to 0.065 M. Under these conditions, D' was linearly related to current density and protein load. Further, log (I)') was linearly related to gel concentration ('ST) ranging from 6 to 14%. However, D' was nonlinearly related to ionic strength. Due primarily to the ionic strength factor, the apparent diffusion coefficient of protein in gels appeared to be approximately IO-fold larger than under the conditions of high ionic strength conventionally used in sedimentation and diffusion studies. Extrapolation of 0' to 0% T, zero protein load, zero current density, and "infinite" ionic strength (assuming noninteraction of these factors), as well as correction for viscosity and temperature, yiefded an estimated free-diffusion coefficient, 1)2o,K.-. of 3.1 x 10-r cm%, which is compatible with previously reported values. These studies indicate that the optimal resolution obtained by PAGE will be considerably lower than that predicted theoretically on the basis of free-diffusion coefficients, and suggest that electrostatic interaction between the proteins and/or deformation of voltage gradient and pH within the protein zones may contribute significantly to band spreading.