Disadvantages of Moving Boundary electrophoresis- The resolution of the technique is very low due to the mixing of the sample as well as over-lapping of the sample components. The electrophoresis technique is not good to separate and analyze the complex biological sample instead it can be used to study the behavior of the molecule in an electric field.

Zone electrophoresis- In this method, an inert polymeric supporting media is used between the electrodes to separate and analyze the sample. The supporting media used in zone electrophoresis are absorbent paper, gel of starch, agar and polyacrylamide. The major advantage of presence of supporting media is that it minimizes mixing of the sample and immobilization of the molecule after electrophoresis. It makes the analysis and purification of the molecule from the gel much easier than the moving boundary electrophoresis. The gel electrophoresis is the best example of zone electrophoresis.

Experiment 13.1 : Analysis of crude cell lysate in SDS-PAGE and determination of molecular weight of unknown protein.

Principle of the technique: In the discontinuous system, negatively charged detergent, sodium dodecyl sulphate (SDS), is used to denature protein and impart a constant negative charge/mass ratio, as a result separation of the protein largely depends on size only. This technique uses 3 different buffers (1) Running Buffer (2) Stacking gel buffer of pH 6.8 (3) Resolving gel Buffer of pH 8.8. Discontinuous gel system concentrate the diluted protein sample into a narrow band, allows application of diluted protein sample. What is the mechanism of stacking protein sample in the discontinuous gel system? Stacking gel buffer is composed of TrisHCl pH 6.8, SDS where as resolving gel buffer contains Tris pH 8.8, SDS and the pore size is large compared to the resolving gel. The mobility of chloride ion presen in the buffer is more than the protein in the sample. The glycine moves slower than the protein sample and as a result protein sample get sandwiched between fast moving chloride ion and slow moving glycine. Due to high electrophoreitc mobility, the protein molecules run fast and stack between the leading and trailing ions. Once protein sample enters into the resolving gel (resolving buffer pH 8.8), glycine ions doesn't stop the migration of protein and protein molecules separate as per their size or molecular weight.