Proteins can be expressed and identified in clones thanks to technology today. The gene enhanced green fluorescent protein (egfp) was cloned into the expression vector pET-41a forming a Glutathione-S-transferase::green fluorescent protein (GST::EGFP) fusion protein. Expression of GST::EGFP was induced by isopropyl–D-thiogalactopyranoside (IPTG) from the plasmid clones. Protein expression was analyzed with Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and western blot. SDS-PAGE separates proteins by size after -mercaptoethanol (BME) breaks the disulfide bones and SDS assures that all proteins have a negative charge. The protein that corresponds directly to GST::EGFP was visualized on nitrocellulose by antibody washes through western blot. The recombinant protein GST::EGFP was purified by a glutathione affinity column where the purification fractions were analyzed through SDS-PAGE. Western blot showed three positive bands being expressed: one for Dr. Blice-Baum’s transformant 8, one for transformant 18, and one for pBIT. Their positive bands lit up purple after the final antibody washes. Eluates 3, 4, and 5 showed a faint band in GST::EGFP fusion protein purification using affinity chromatography around 59 kilodaltons (kDa). This properly met the weight of the fusion protein. The quantified amount of purified GST::EGFP fusion protein was unable to be determined through fluorescence assay due to lacking a microplate reader containing a UV illuminator detector.
Specific proteins can be isolated and identified in clones with the help of technology. Technology has helped increase the knowledge in the field of molecular biology immensely. Having tools available to us such as SDS-PAGE, western blot, and affinity chromatography we can learn more about proteins and how they play a role in biochemistry, cell biology, immunology, molecular biology, virology and much more. SDS-PAGE contributes to the separation of proteins based on size. Before having this technology, protein separation based on charge had to be done on paper followed by columns of sucrose. Now, protein can be separated based on size on polyacrylamide gel thanks to a Massachusetts Institute of Technology graduate student’s discovery in 1964. He discovered that SDS can split E.coli’s proteins to increase their electrophoretic resolution when polyacrylamide gel contained SDS (Pederson, 2007).
SDS-PAGE is fast, cheap and easy to use, leaves room for little error when inserting the dye into the wells and gives a simple reading for the protein’s molecular weight. Ulrich Laemmli’s method for SDS-PAGE with polyacrylamide is a widely used method by many in molecular biology. In addition to SDS-PAGE being able to analyze recombinant protein expression and being used in purification techniques, it can also determine molecular weight and protein heterogeneity in diagnosing hereditary red cell membrane disorders (Rath, Cunningham, & Deber, 2013).
SDS-PAGE ran on polyacrylamide gels are discontinuous protein gels whereas agarose gels are continuous gels. Each have their advantages, disadvantages, and can be used for different reasons. Pore sizes and the electric fields on each of these gel matrices affect how DNA and proteins migrate on each. SDS-PAGE shows better resolution of the protein bands. DNA separation and migration occurs on agarose gels since DNA molecules are larger migrate much slower on polyacrylamide gels. Proteins are run on polyacrylamide gels since they are smaller and polyacrylamide gels have smaller pore sizes (Stellwagen, 2010). SDS and -mercaptoethanol (BME) are added to polypeptide sequences. SDS assures that all proteins will have a negative charge and then BME breaks covalent disulfide bonds (Carson, Miller, & Witherow, 2012).
Western blot can be used to separate and identify certain proteins from a mixture of proteins removed from cells, just like identifying a fusion protein from GST::EGFP. It was developed by Edwin Southern in 1975 who also developed southern blotting (Mahmood & Yang, 2012). Western blotting separates proteins by gel electrophoresis, transfers the proteins to a nitrocellulose membrane to a blot, probes the blot for the protein of interest and can then detect the protein through fluorescence (Signore & Reeder, 2011). This is used in biomedical research to extract proteins from tissues or cells. Western blot utilizes Ponceau staining to visualize the total protein band for samples. It can be done on a few different membranes, including nitrocellulose membranes. Ponceau staining has a negative charge so it can bind to amino groups of protein with positive charges (Ponceau-S Stain, 2016). The protein bands should stain red when binding to the proteins.
Western blots are blocked to prevent non-specific binding with antibodies. Blots are incubated with antibodies. There is direct detection and indirect detection. Direct detection only uses one antibody to detect the specific antigen on a blot but has many disadvantages. Indirect detection uses two antibodies and is much more favorable. The primary antibody is detected by a conjugated secondary antibody. Secondary antibodies can be labeled with biotin, fluorescent probes, fluorophores, and enzyme conjugates to allow more options for multiple detection methods (Overview of Western Blotting, 2016).
There are monoclonal and polyclonal antibody probes. A monoclonal antibody probe is used to bind only to one antibody species at a specific site whereas a polyclonal antibody probes can bind to different sites of the desired molecule. Monoclonal antibodies only recognize one epitope in an antigen. They can be produced from a B cell of an immunized mouse’s origin to create a population of antibodies that are all identical to each other. Polyclonal antibodies are collected from various B cell clones that have been stimulated by the immune response of an immunized animal such as a goat, sheep or rabbit (Monoclonal & Polyclonal Primary Antibodies, 2017).
Affinity chromatography is a quick and easy way to separate and purify recombinant proteins. Proteins are purified from inhibitors, protease contaminants and protected from denaturing (Cuatrecasas & Anfinsen, 2003). With affinity chromatography cell lysate is used; it binds to a biospecific ligand to the molecule of interest. An example of this would be a GST ligand binding to glutathione. The sample with proteins containing the fusion protein of interest would be injected into the affinity column and then the molecules with affinity for the ligand would be adsorbed. Impurities get washed form the column and then the target molecule is eluted. After the elution, the protein of interest will be left in its purest form (Carson, Miller, & Witherow, 2012).