Question

By gel electrophoresis, how do you know if your protein binded to the column? How would you modify the purification next time to make it more efficient?

Answer 1

Separation and purification of molecules, like nuclei acids, lipids, or proteins, is mainly based on the differences in their physical properties. Proteins may vary according to the:

a) Amino acid sequence and chain length. b) Size, in terms of length or mass. c) Three-dimensional structure or spatial arrangement. d) Net charge, determining hydrophilic or hydrophobic nature.

The process of electrophoresis is based on separation of molecules due to difference in charge: mass ratio. Due to various three-dimensional structures, rigidity, effect of di-sulfide bonds, or protein existing as complex (like oligomers), protein denaturation is required. This may be done by heat, detergents like Sodium Dodecyl Sulfate (SDS) or Beta-mercaptoethanol.

For column electrophoresis, the gel is separated into three portions with: a) Separating or running gel, b) stacking gel, and c) Sample gel.

Two important processes are 1. Nickel-column electrophoresis or 2. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

After the gel is run with the protein samples, to detect the proteins, methods that may be applied are:

1. Coomassie blue – it stains proteins non-specifically with bound proteins.

2. Western blotting- using antibodies labelled or tagged with fluorescent dyes, radiolabeled antibodies. These will bind the protein sample and can be detected as per the tag used.

3. Use of histidine tags- Six (or nine) histidine residues are incorporated as poly-histidine tags, for purification of recombinant proteins. His-6 tags can be introduced in -terminal or C-terminal of the protein. They can be easily detected due to the reason that, several immobilized metal ions (like nickel, cobalt, copper).

The method of protein purification, elution of proteins may be enhanced by adding imidazole.