Western Blot Analysis of Serum Proteins to Determine Evolution of Proteins

Purpose: To investigate the evolution of proteins by using the Western Blot analysis of serum proteins.

Procedures:

Electrophoresis:
The purpose of the electrophoresis of the serum proteins was to compare the electrophoretic bands of the proteins to correlate with the latter antibody reaction. The protein samples must be boiled to be denatured, thereby unfolding the proteins for electrophoresis.

Western Blot - Preparation of Blot:
The gel must be redone without the staining because the nitrocellulose membrane is not supposed to have any staining on it. The "sandwich" is set up in such a way to allow osmosis to carry the proteins from the gel to the nitrocellulose paper. The two sheets of blotting paper soak up the transfer buffer. As the blotting paper becomes saturated, the transfer buffer slowly moves into the agarose gel, through the nitrocellulose membrane and into the final two sheets of blotting paper which prevents reverse osmosis. As the transfer buffer goes through the agarose gel, the proteins are being picked up. However, they are trapped by the nitrocellulose membrane from further upward movement.

Staining:
The protein bands on the nitrocellulose membrane must be stained so that the interactions with the antibody will be visible. Gelatin solution is used to cover the gel for several reasons. Gelatin itself is able to preserve the proteins because it lacks essential amino acids to replace the proteins. Therefore, the gel should be able to be stored for up to a week without decreased efficiency of results. Furthermore, gelatin's insolubility in organic solvents adds to its effectiveness.

Antibody Reaction:
The antibody to cow albumin is incubated with the nitrocellulose membrane to produce a reaction between the antibody and the albumin in the serum proteins. The stronger the interaction, the greater the similarity to cow albumin. The Color Development Solution allows the antibody reactions to be visualized.

Data from Biology Workbench:

The unrooted phylogenic tree of myoglobin for cow, horse, goat, sheep, donkey and chicken was found using Biology Workbench.