Protein Extraction and Bradford Assay Quantification

Tissue Homogenization and Protein Extraction

Tissues are rich in proteins that fulfill different functions and therefore possess different characteristics. Depending on these traits, it is possible to solubilize, separate, and purify them through various methods.

Methods for Cell Disruption

The first step in the extraction of most proteins and subcellular structures is the disruption of tissues or cells to obtain a cell lysate. In this lysate, the protein or multiprotein complex subcellular structure is kept in conditions that permit isolation. This is accomplished using procedures commonly known as soft mechanical homogenization.

These methods produce the rupture of cell membranes gently, thereby releasing the cellular contents. Common techniques include:

• Osmotic shock
• Mortar (friction)
• Sonication

The obtained proteins must be solubilized in a buffer that helps extract them from the cells and maintain them for subsequent use. The aim of this practical session is to perform a protocol for tissue homogenization and the extraction of total protein for further quantification and characterization.

The Bradford Method for Protein Quantification

One of the most common operations used in research laboratories is the determination of the total concentration of proteins present in a preparation. The Bradford method is widely used for rapid and accurate determination of total proteins in cell fractions and concentration analysis for use in electrophoresis gels.

Principles of the Bradford Assay

The standard test has a sensitivity from 0.1 to 1.4 mg/mL of protein. The test is based on the observation of the maximum absorbance change of the acid solution Coomassie Brilliant Blue, ranging from 465 to 595 nm when binding occurs with the protein.

This union is due to the stabilization of the anionic form of the dye by hydrophobic and ionic interactions, causing a visible color change. The dye has a large number of nonpolar groups but has two polar groups (SO₃^2-) that bind to positive charges of amino acid side chains, specifically arginine or lysine, which is a known disadvantage for the method.

Calibration and Standards

This test also requires the completion of a calibration curve for the determination of protein concentration in a sample, commonly using BSA (Bovine Serum Albumin) as the standard protein.