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Purification of GFP Protein, Research Paper Example

Pages: 7

Words: 1864

Research Paper

Abstract

Green Fluorescent Protein (GFP) usually emits bright green fluorescence light when it is exposed to blue or ultraviolet light. The protein has been used as a marker for the gene expression visualization, protein localization in both living and fixed tissues. It is also used for protein targeting in organisms and intact cells. The direct purification method was designed to purify the recombinant GFP from the intact Escherichia coli by using a preparative polyacrylamide gel electrophoresis (n-PAGE) via a continuous buffer system. The current experimental study comprised of the quantification of protein concentration (Bradford Assay). When carrying out biomedical experimental research, it requires the measurement or protein concentration in samples of solutions. Several techniques have been developed despite the fact that they have several limitations. For example, some of the methods used are not sensitive enough or depend on the reactions with specific amino acids in the proteins. Further, some amino acids have contents that vary. Hence no single Bradford essay is suitable for all proteins. In the experimental study, the procedure used was based on the binding of a dye to protein in acidic medium to cause a change in the wavelength of the maximum absorption. The assay requires only a single reagent, which produces color development. A sample is prepared using a generic protein to determine the total concentration of a protein. An absorbance was used to determine the intensity of the protein of an unknown sample. The relationship between the absorbance and the concentration of the protein was established by creating a standard curve through the application of linear regression. Further, the standard curve was generated by plotting the absorbances derived from the known concentration of the protein. Several dilutions of the unknown sample of the protein were prepared to obtain an absorbance, which could fall within the required linear range of the standard curve.

Introduction

Denaturing SDS-PAGE is a standard technique that is the same as the agarose gel electrophoresis since the electrical field is placed across a gel pores of varying sizes. The molecular chain molecules migrate in the same direction and get separated by their molecular weight. In this context, the gel is a polymerized acrylamide and a mix of bis-acrylamide. The long chains that travel through are amino-acids, and their sizes are estimated by making comparisons of the unknown protein samples to a ladder that has a standard molecular weight linear peptide chains.

Proteins are made up of several strands of amino acids. They have different properties and charges. Some of the amino acid chains are positively charged, negatively charged, or neutral. Therefore, depending on the type of amino acid that is involved in the process, any protein has a charge, which may be positive, negative, or neutral. Further, proteins often assume several layers of additional structure, despite the fact that they are made up of linear polypeptides of several chains of amino acids joined together, which forms their primary structure. The secondary structure mainly takes place as a result of hydrogen bonding, and the two main types are the alpha helix and the beta pleated sheet (Hazlin 7). The tertiary structure occurs when a protein folds to ensure that they are maximally stable at the lowest energy state. Thus, the tertiary structure depends on the hydrophobicity, flexibility, as well as the formation of disulfide bonds between the cysteine residues and the hydrogen bonding that occurs between other side chains. Further, the quaternary structure takes place when there are several proteins joined together to make a complex structure.

Running a native protein through a gel would be problematic, especially when an electric field is put across.  Although native proteins have several applications, the standard practice is to eliminate the second degree, third degree, and fourth-degree structure and to impose the same charge on all the resulting polypeptide chains. The process of removing the protein structure is done by denaturing the sample before loading. The protein samples are mixed with denaturing agents are heated to a boiling point before being loaded to a gel. Therefore, the high temperatures in the presence of the denaturing agents and a stronger SDS detergent assist in breaking the hydrogen bonds and disulfide bonds.  Eventually, the process turns the proteins into long strings of amino acids. The presence of a negatively charged SDS in a sample buffer coats the proteins with a uniform negative charge. The process masks the intrinsic charges on the side groups of the amino acids. SDS also binds reasonably consistent to the linear proteins meaning that the charge of the protein will be approximately proportional to its molecular weight. Furthermore, SDS ensure that the proteins remain coated through the process. The critical factor that determines an SDS-coated protein is the molecular radius, and since they have the same charge to the mass ration, there will be no differential migration depending on the charge. As mentioned, proteins can move through a gel depending on their shape and structure. The degree of separation can be adjusted by changing the concentration of the acrylamide applied and the size of the pores. During the separation process, smaller proteins of interest are separated better by higher levels of acrylamide.

Another critical approach to denature proteins involves the use of a gel and a buffer system. When using this approach to denature SDS PAGE electrophoresis, a gel is run vertically coupled with the use of a discontinuous buffer system, implying that the buffer in the gel and the tank are dissimilar. There two types of gels used in the process are stacking gel with a low acrylamide and a separating gel a separating gel that has a high acrylamide. The two serve different purposes. For instance, the wells are developed in the stacking gel and the process, the denatured sample is compressed into a thin line by the stacking gel before entering the separating gel. Ostensibly, the separating gel is the place where the differentiation occurs due to the differences in the mobility based on size. The gels can be blotted on a membrane that can retain the pattern of the protein bands. The sample is then probed using an antibody against the protein or a tag attached to it. Consequently, secondary antibodies that have enzyme activity are used to observe the proteins detected through a process known as western blotting.  Protein tags refer to the addition of something to a protein to detect or tract it. Most tags are added through modifying the gene products either at the beginning or the end of a protein polypeptide chain. Individual tags are small strings of amino acids that are added at the end of a protein. Since linear protein tags do not form secondary structures, they can readily get purified. Larger protein tags with secondary structure can help in making a sample soluble, but when denaturation is necessary, they would not be appropriate for binding.

Background

Electrophoresis is the movement of charged particles within an electric field, the process is by the charge and voltage, the distance between the electrodes, as well as size and shape of the molecule. Other factors that determine the movement of particles include temperature and the duration taken. Notably, the process takes many forms, which are agarose gel electrophoresis and polyacrylamide electrophoresis. The experimental study used polyacrylamide electrophoresis, which comprises of polymers acrylamide that is cross-linked with acrylamide to form a porous medium with the consistency of firm Jell-O. Other substances that are added to the mixture are ammonium persulfate and TEMED. When ammonia persulfate reacts with water molecules, t breaks down and form free radicals. The free radicals trigger a chain reaction when in a solution of acrylamide and bis-acrylamide. The free radicals of the two substances are formed, which enhances the polymerization process to a stage of completion. Consequently, the long polymers produce a viscous solution. After adding bis-acrylamide, the long chains of acrylamide are cross-linked, which results in a gel that has an average number of cross-links per volume. The process produces pores of a given average size. The mobility of a protein is determined by the charge, shape, as well as size. Therefore, provided that the samples are treated to have a normal charge and are unfolded, the electrophoretic movement of the protein will largely depend on size.

The experimental determination of the of the molecular weight for the de-natured proteins will be complicated. However, the process can be done. During the experiment, sodium dodecyl sulfate (SDS) is used to bind the proteins in a constant ratio of 1,4 g SDS per each gram of protein. As a result, it covers the protein with negative charges; hence overwhelming the intrinsic charge and affects the secondary, tertiary, as well as quaternary structure to produce a linear peptide chain. However, the availability of a reducing agent assists in protein denaturation. The process reduces all the disulfide bonds. Similar net charge and shape characterize all proteins. Therefore, application of an electric field to the gel will cause the proteins to migrate towards the positive electrode at a rate relative to the molecular weight. For this reason, two proteins with the same molecular weight will exhibit the same migration pattern regardless of the amino acid sequence or a native structure. Also, as the mixture of the protein moves through the gel, the movement of the larger proteins will be slowed compared to the smaller proteins. The extent of retardation depends on the size of the pores. The difference in the migration rates causes the proteins to get separated into discrete bands depending on their molecular weight. For instance, heavy glycosylation will eventually change the apparent molecular weight.  Therefore, it is essential to understand that a band on a gel represents proteins that have the same molecular weight. Similarly, a single band does not equal a pure population of a particular protein.

The standard process used to separate molecules is known as chromatography. There are several types of chromatography, but all of them depend on the same principle. Thus, the basic premise of the process ensures that the molecules to be separated from the mixture will interact with both mobile and immobile phases. The process depends on the chemical and physical characteristics of the molecules to be separated. Gel exclusion chromatography enables molecules in a mixture to be separated based on their sizes. It comprises a porous gel matrix, which is chemically inert and the molecules to be separated do not interact with the buffer. Adsorption chromatography entails interaction between the molecules to be separated and the porous gel matrix. When using this approach, the porous gel matrix has the chemically inert support that has chemically reactive moiety bonded to it. The group can be a molecule with a positive or negative charge, which involves an exchange of ions. The other type is hydrophobic interaction chromatography, and when using the technique, hydrophobic proteins tend to behave well by binding the sorbent and elute with the change in salt concentration. In affinity chromatography, the interaction between a molecule and the matrix are more specific compared to the charge. The technique can theoretically allow for a single-step purification, provided that the affinity ligand is specific.

Works Cited

Hazlin, Mohamed. The Effect of Buffers Ph on Electrophoretic Purification of Intracellular Green Fluorescent Protein. Kuantan, Pahang: UMP, 2014

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