Proteins

Overview

Proteins were first discovered in the 18th century by biologists who were exploring the components of cells. Researchers extracted these molecules and then observed the physical changes that happened when they were added to acid or heat. For example, one of the first proteins identified was albumen, which is present in the clear, viscous liquid that we call the white of the chicken egg. When heated, however, the egg white hardens and changes color to become opaque and white.

Each protein is a polypeptide, which means that it is a string of amino acids that are connected to one another through chemical linkages. Through their physical and chemical properties, each amino acid in the polypeptide chain influences the shape and charge of the protein. This allows the polypeptide chain to twist and fold into a three-dimensional structure that defines its function. Furthermore, some proteins bind with one another to create complexes that perform specific functions.

So how do we explore proteins in the biotechnology laboratory? One of the most effective ways to study a protein’s structure is using polyacrylamide gel electrophoresis, or PAGE. This technique uses electricity and a porous gel matrix to separate mixtures of proteins into discrete zones, or bands, based on their physical properties. This includes the molecule’s charge, its shape, its size, and the number of subunits in a protein complex.

Proteins pose a unique challenge for electrophoresis because they have complex shapes and different charges which affects how they migrate through the gel. To accurately separate proteins by molecular weight and not by shape or charge, protein subunits must be disconnected and the secondary structure of the protein must be unfolded. This is accomplished using the anionic detergent sodium dodecyl sulfate (SDS), a reducing agent, and heat. Heat disrupts hydrogen bonds, allowing for the unfolding of proteins. The SDS molecules stick to the protein, negating its inherent charge. The reducing agent breaks covalent bonds that link protein subunits.

After denaturation, the mixture of proteins is added into depressions (or “wells”) within a gel, and then an electrical current is passed through the gel. Because the SDS-protein complex has a strong negative charge, the current drives the proteins through the gel towards the positive electrode. At first glance, a polyacrylamide gel appears to be a solid. On the molecular level, the gel contains channels through which the proteins can pass. Small proteins move through these holes easily, but large proteins have a more difficult time squeezing through the tunnels. Because molecules of different sizes travel at different speeds, they separate into discrete “bands” within the gel. After the current is stopped, the bands are visualized using a stain that sticks to proteins.

"Edvotek at Home" is a set of resources to teach the basics of Edvotek’s labs through worksheets and presentations. While we believe in the importance of hands-on learning, these free online learning tools are ideal if you can not perform the hands-on experiments in class. Each set includes a student sheet, an instructor’s guide, and an accompanying powerpoint presentation and results sheet. This resource is provided in a downloadable zipped folder below.

Protein Electrophoresis - Proteins produce a unique challenge for electrophoresis because they have complex shapes and different charges, which affect how they migrate through the gel. SDS polyacrylamide-gel electrophoresis, or SDS-PAGE, is a technique that is used to separate proteins according to their molecular weight. The protein samples have been denatured by incubation with a strong detergent, then loaded into a polyacrylamide gel. An electrical current is passed through the gel, pushing the proteins through the gel towards the positive electrode. Since molecules of different sizes travel at different speeds, they separate into discrete “bands” within the gel. In this experiment, SDS-polyacrylamide gel electrophoresis is used to develop an understanding of protein structure, function and diversity.

These short courses couple theory with active experimentation to help you update your skills and knowledge in various areas of biotechnology.

Using Biotechnology to Diagnose HIV/AIDS - The Human Immunodeficiency Virus (HIV) causes acquired immune deficiency syndrome (AIDS), a serious disease that suppresses a patient’s immune system, leaving them susceptible to infections. In this simulation, we’ll perform two common tests (western blot, ELISA) used by doctors to diagnose an HIV infection.

A Bright Idea: Using GFP to Teach STEM - Bring exciting STEM learning techniques into your classroom laboratory! In this hands-on workshop, we will build a size-exclusion chromatography column. The column is used to purify green fluorescent protein (GFP) from a crude bacterial extract. Proteins containing fractions are identified by fluorescence and analyzed for purity by SDS-PAGE.