Electrophoretic Separation Of Proteins Biology Essay
Proteins are vital in nearly everything organisms do chemically, structurally, and physically. This experiment was designed to separate proteins for analysis through molecular weight in order to characterize and identify an unknown. The four distinguishing molecular characteristics of proteins used for separation included unique molecular weight (size), charge and affinities, conformation, and length (Carpenter, et al, 2008). Specifically, this experiment was designed to pinpoint a species substitution between imitation crab meat and real crab meat utilizing the technique of gel electrophoresis. After staining the gel, one must look for concordance in migration between any two samples to correctly identify the unknown and determine if a species substitution occurred. Because the imitation crab meat, which is mainly Pollack fish, is like one big muscle, it is hypothesized that there will be an abundance of actin and myosin highlighted by two distinct bands on the gel. Two 15 µL samples were independently isolated from the imitation crab meat, real crab meat, and those of the unknown, all of which were loaded on the gel together and studied in detail. Characterization by this biochemical test indicated that the unknown sample was indeed that of real crab meat. The electrophoresis banding patterns differentiated the isolates based on molecular weight illustrating that the unknown and real crab meat samples migrated to nearly the same location on the gel. It is concluded that there is no qualitative difference between the unknown sample and the real crab meat and that there was not a species substitution.
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Proteins account for more than 50% of the dry mass of most cells (Reece, 2005), and they range in functionality. From acting as a catalyst or an enzyme to helping protect against disease, proteins are one of the central macromolecules of life. The function of a protein is determined by its shape. The shape of a protein is determined by its primary structure or sequence of amino acids. Scientists have used separation techniques, collectively known as chromatography, to isolate a protein of interest. Gel electrophoresis is a very efficient tool for exploring these differences. This technique separates macromolecules on the basis of their rate of movement through a gel in an electrical field based on size, electrical charge, and other properties (Carpenter, et al, 2008). Due to the muscular nature of fish, it was hypothesized that the bands of the imitation fish would be double stranded by nature, representing actin and myosin. In addition to that, the real crab meat would have more bands because it has more classes of proteins. It is also predicted that whatever lines up distance wise with the unknown sample is the correct identification of the sample itself. Procedurally, the sample is run in a matrix or meshwork of pores such as an agarose gel, which is utilized to separate the proteins (Merril, 1998). If you increase agarose percent, then pores will be smaller (Merril, 1998). This directly results in poorer separation because molecules have a greater likelihood of getting stuck. The distance a molecule travels while the current is on is inversely proportional to its length (Carpenter, et al, 2008). In other words, the shortest molecules end up in bands at the bottom of the gel. Sodium dodecyl sulfate (SDS) is the most popular system implemented to separate proteins based on size differences. This compound is an anionic surfactant that is widely used as a detergent (Carpenter, et al, 2008). Its main functions include giving the protein more negative charge to facilitate migration and denaturing the protein. In fact, SDS inhibits gel polymerization as well by carrying out these vital functions (Merril, 1998). As described herein, results demonstrate that the unknown sample was indeed real crab meat through electrophoretic separation of proteins.
In one earlier study, a molecular approach similar to the one described above was employed to analyze the genetic diversity of complex microbial populations. This technique is based on the separation of polymerase chain reaction-amplified fragments of genes by denaturing gradient gel electrophoresis (DGGE) (Muyzer, 1993). DGGE analysis of different microbial communities illustrated the presence of up to 10 distinguishable bands in the separation pattern, which were rooted from a variety of species that were once part of a population (Muyzer, 1993). This proved that it was possible to correctly identify species which comprise only 1% of the total population (Muyzer, 1993). The results obtained through both techniques signify its practical uses. Not only do these techniques aid in exploring genes of interest but also helps to conceptualize genetic diversity as well. This experiment along with the primary experiment emphasizes not only the value of these techniques, but also their real world applications as well.
MATERIALS AND METHODOLOGY:
First, a 1.0 g sample of either real crab meat or imitation crab meat was provided. This was placed in a mortar that contained 1.0 ml of sample preparation buffer, composed by SDS and a specific dye. The charge of the protein was 1.4 g SDS/g of protein. The mixture was grinded using the pestle for about ten minutes. An important observation made within the group was that the sample was not yet fully liquefied after the stirring process, possibly skewing the outcome of the experiment. Next, the samples were centrifuged for 5 minutes. After centrifusion, 0.5 ml of the sample was to be pipetted into a 1.0 ml micro centrifuge tube. Because our supernatant was not fully liquefied and very limited, only about 0.3 ml was actually pipetted from our real crab meat sample. However, 0.5 ml of the imitation crab meat was retrieved from the other group. All tubes were labeled accordingly. An unknown sample was then given. A hole was poked in the lid of each tube to allow air to escape during the 3-minute 95°C water bath that proceeded. The gel electrophoresis chamber and agarose gel was pre-prepared. There are eight wells (starting from left to right), 15µL of the real crab meat was loaded into wells 2 and 3, 15µL of the unknown sample was loaded into wells 4 and 5, and 15µL of the imitation crab meat was loaded into wells 6 and 7. Notice that wells 1 and 8 were left vacant. During the pipetting, one of the group members had difficulty inserting the imitation crab meat sample into appropriate wells therefore eventually affecting band clarity. Electrophoresis was carried out at a constant current of 250 milliamperes at a voltage of 85 volts for approximately 30 minutes. Once the 30 minutes expired, gloves were worn and the gel was then appropriately placed in a staining tray filled with Coomassie Blue staining solution for 15 minutes. The gel was then destained for 30 minutes with destaining solution. The gel was finally observed using a light box, paying particular attention to the distances of migration, band intensities, and relationship between the three bands collectively. Determine if the unknown sample is an imitation or real based on these observations. Also, determine some structural components of each sample based on what was observed. A picture was taken just in case further analysis was needed. A sketch was then drawn and results were recorded.
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Upon first glance at the gel using the light box, it was extremely hard to distinguish band lengths and underlying characteristics. However, upon further review, it was observed that the real crab meat and the unknown sample (wells 2-5) matched up almost evenly. Next, the false or imitation crab meat sample had two distinct bands and was visually unlike the real or unknown. The number of bands observed in the real and unknown samples were quite vague due to random error made in setting up the gel. In addition to that, the band intensities for the true and unknown samples were also more pronounced than the imitation crab. As far as migration was concerned, not only did the real crab meat and the unknown sample travel relatively the same length across the gel, but also traveled further than the imitation crab meat. Overall, the gel electrophoresis was performed very accurately considering only two groups received an image (with my group being one).
Figure 1. Pictoral SIDEVIEW Representation of Banding Patterns Using A Light Box. If the gel wells were facing upwards, it would be labeled as TTUUFF (Wells-2-7) starting from left to right. T represents real crab meat (true), U represents unknown, and F representing the imitation crab meat (false). Protein Gel Electrophoresis with a voltage of 85 volts and a current of 250 milliamperes for 30 minutes was utilized to generate this picture.
Figure 2. Hand-drawn Schematic TOP View of the Gel. All details that are not relevant to the results were omitted to convey band number in each of the three samples, the intensity of the bands, and how far they have migrated. Each sample is labeled above.
The results did somewhat support the hypothesis in that after electrophoretic separation of the proteins, the imitation crab meat had two distinct bands, whereas the real crab meat and the unknown had more. The two distinct bands do in fact represent the two main proteins found in muscle: actin and myosin. Since it was determined that the real crab meat band migrated the same distance as the unknown, these results may suggest that not only is the unknown sample real crab meat but also suggests similar molecular weights as well. Since these two samples migrated further than the imitation crab meat and ended up in bands at the bottom of the gel, these molecules are said to be the shortest. Although this is somewhat unusual, one must remember that the imitation crab meat also has proteins and other compounds that may establish it as a longer molecule.
These results are vital in that they relay to a consumer the power of scientific techniques to expose molecular impurities. For other scientists, gel electrophoresis is utilized to provide genetic information in a wide range of bio-chemical fields. For instance, DNA can be analyzed instead of proteins for legal reasons to prosecute criminals, to detect genetic diseases, and to test for paternity (Gel electrophoresis, 2006). Taxonomists would also take special interest in this technique to distinguish key characteristics between various species (Gel electrophoresis, 2006). One especially interesting use of gel electrophoresis is the detection of a sickle cell trait or sickle cell anemia. The presence of ßA and ßS alleles is determined by restriction end nuclease digestion of a probe (Saiki, 1985). The genotype can then be determined. Although hemoglobin S (sickle) and hemoglobin A (normal) differ by only one amino acid, they can be easily separated using gel electrophoresis (Saiki, 1985). If both types of hemoglobin are identified, the individual is a carrier of the sickle cell trait (Saiki, 1985). If only hemoglobin S is present, the person most likely has sickle cell anemia (Saiki, 1985).
Random errors are almost always present in nature. In this particular experiment, although our group obtained a gel with bands, many errors may have disturbed precision. In the very beginning, the real crab meat was not fully grinded to liquid form. This will definitely affect band intensities and formation. Next, the imitation crab sample was not prepared by our group. Anything pre-prepared always runs the risk of endangering precision. Furthermore, most importantly, when imitation crab meat was loaded into wells 6 and 7, the entire 15µL was not loaded due to inexperience with handling an agarose gel. Thus, the results are not entirely reliable since too many factors and variables were overlooked.
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Although the data presented is not contradictory with any previous studies, the methodology somewhat is. Agarose is most commonly used to separate large macromolecules such as nucleic acids. A polyacrylamide is more commonly used to separate proteins, however (Merril, 1998). Polyacrylamide gel electrophoresis (PAGE) is an optimal method for characterization of protein molecules on the basis of size, conformation, purity, and charge (Merril, 1998). Proteins migrate to an electrical field through pores in a polyacrylamide gel matrix, and as acrylamide concentration increases, pore size decreases (Merril, 1998). Additionally, the type of gel-electrophoresis that was utilized was one-dimensional. Although this was appropriate and more affordable, two-dimensional gel electrophoresis is a widely employed method for the analysis of complex protein mixtures extracted from these meats. This technique separates proteins in two different steps according to two unique properties (Alban, 2003). The first step focuses on separating the proteins according to their isoelectric points (pI) (Alban, 2003). The second step is similar to one dimensional electrophoresis in that SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is utilized, which separates proteins based on their molecular weights (MW) (Alban, 2003). Without being too heavily invested in the methodology, this technique will allow you to compute so many different characteristics between two different molecules. It would have been interesting to place the unknown sample, which we found to be real, and the imitation crabmeat sample together and observe distinguishing characteristics between each. Furthermore, it is widely held that molecules are more effectively separated in 2-D electrophoresis than in 1-D electrophoresis as well (Alban, 2003).
Because of the drastic similarities present between the unknown sample and the real crab sample, it can be concluded that there was not a species substitution after all. However, human and experimental error was overwhelming. Future research needs to be conducted incorporating more samples and more gels. Also, another study needs to be conducted that looks more heavily into the structural and physical similarities between these two meats.
In conclusion, data collected from this research does somewhat support the initial hypothesis. Due to the vast amount of other variables, conflicting conclusions of this experiment with others, and human and experimental error, more trials and larger sample sizes need to be utilized to fully visualize these assertions. Overall it was interesting to use gel electrophoresis and put it to practical use.
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