Separation of Proteins and Determination of Species Substitution

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Table of Contents

Abstract
Introduction
Materials and methods
Results
Discussion
References

Abstract

This experiment was aimed to solve the case of species substitution, in particular, it compared three samples, one of which was real crabmeat, the second one was an imitation, and the third one was not identified. Gel electrophoresis has been used to test this unknown sample. The proteins were separated according to their molecular weight. This empirical research demonstrated that the banding patterns of real and unknown samples coincided with one another, whereas the proteins of the third (fake) sample migrated differently. Apart from that, this study showed that the determination of species substitution normally requires the combination of various research techniques such as near-infrared spectroscopy and immunological analysis. This eventually enables us to get more accurate results. This experiment has been beneficial to the extent that it provided deep insights into the peculiarities of chromatographic procedures, their limitations, and the guidelines which should be followed in order to attain accuracy.

Introduction

The experiment that has been carried out was aimed at determining the so-called species substitution. In particular, it was necessary to ascertain whether the unknown sample (allegedly of crabmeat) was real or imitation. For this purpose such a method as gel electrophoresis was used to separate proteins. The main task was to charge molecules and trace their migration patterns. On the whole, similar studies have already been conducted by other researchers, for example, by Javier Gayo et al (2006), who investigated the adulteration of crabmeat by means of visible and near-infrared spectroscopy. Furthermore, a group of authors under the direction of Elena Dominguez also investigated species substitution by using immunological techniques. This problem attracted so much attention because such unlawful practices pursued by restaurant owners can greatly imperil the health of the visitors, as some of them may have an allergy to fish or other substitute products. Although it is too early to jump to any conclusion, it is possible to presume that the proteins of a fake sample will travel slower than real ones due to the differences in molecular weight. The next section will detail the specific procedures employed during the experiment. It is also of crucial importance to discuss this experiment in connection with other works, devoted to the study of protein separation and species adulteration. It is necessary to understand whether they are in league with one another or not. The key objective of this report is to understand how this experiment is related to such areas of study as chromatography and food forensics.

Materials and methods

As has been mentioned before, gel electrophoresis was used as the main research technique. Overall, it relies on the movement of ions in an electric field (Garret & Grisham, 149). The proteins were placed in a solution that contained sodium dodecyl sulfate and a dye. It should be borne in mind that sodium dodecyl sulfate performs the following functions: denaturation and separation of proteins according to their molecular weight (Scheppler et al, 163). Therefore, one can assume, that the proteins of the unknown sample if is a fake one would travel either faster or slower than those proteins of the real one. But one should take into account that the findings of this experiment will be valid under certain conditions namely that samples are not contaminated, that appropriate temperature is maintained, and that the gel contains a sufficient percentage of agarose. Thus, necessary precautions should be taken in order to meet these standards.

There are some other criteria according to which one can distinguish fish from crabmeat. Fish as a species have an increased level of antibodies or gamma globulin proteins in their bodies and this is one of the ways to detect substitution of crabmeat. (Hall, 165; Dominguez et al, 280). However, for this purpose, one should apply a set of immunological forensic techniques that are widely utilized in the field of food research. Besides, we should set stress on the idea that this is a blind experiment, which implies that the researcher is unable to predict the results of the investigation. There is a great likelihood that the unknown sample is similar to the real crabmeat and there is no reason for panic.

Therefore, each of the three samples (real one, imitation, and unknown) were placed in a mortar with 1,5 ml of sample preparation buffer, and then the mixtures were ground with the help of a pestle. Afterward, they were poured into a 15 ml centrifuge in order to pellet the solids. Then 15 μl of each sample were loaded into the wells. The electrophoresis procedure lasted for about half an hour. The apparatus was run at 170 volts (Carpenter et al, 2007). Subsequently, the gel was covered with a destaining solution and this enabled us to see the banding patterns. Given that the charge is the same, the only factor that affects the migration of protein is the molecular weights. The smaller protein would travel faster than a larger one. Nevertheless, one should not forget that degradation of samples or overconcentration of polyacrylamide may change the speed of migration.

Theoretically, other determinants influence the mobility of a protein, namely, their charge on their native form but due to the use of buffer this effect is reduced to a minimum. It should be pointed out that the charge of the buffer is never equal to that of one of the proteins (Stanley, 183). Furthermore, if these samples were run on a gel with a higher percent of agarose, the whole process would be showered and it will also change the distance between the bands. The increased percentage of agarose is more suitable for other types of electrophoresis. These are the hypothetical limitations of gel electrophoresis, yet, it seems that all of these pitfalls were avoided in the course of the experiment. So, the findings that we have obtained can be considered valid.

Results

Judging from the results of this investigation, one can say that the observed banding pattern of the real sample and that one ones the unknown one were similaoneso one another. The first five lanes (2 lanes belonging to a real sample, and three lanes of the unknown one) traveled the same distance. Most, importantly, these five lanes traveled further than the last two lanes (imitation sample).

Discussion

Given these finds, it is possible to arrive at the conclusion that the unknown sample, which has been provided to us, is real crab meat. These two samples showeda similar banding pattern due to the fact that molecular weight of the proteins was the same. In turn, the proteins of an imitation sample had a lesser electrophoretic mobility. In the previous sections, we have marked out factors that can adversely affect the accuracy of the results, namely, the net charge of the proteins, the percentage of aragose but this experiment was carried out in accordance with requirements, set for gel electrophoresis. This study demonstrates as a method of protein separation gel electrophoresis can prove useful in such field as seafood forensics.

Still, it has to be acknowledges that the results of this investigation cannot be taken as very important only by themselves. They should be viewed only as a part of a large field of study such as food forensics. As a matter of fact, the efficiency of gel electrophoresis as a method of separating proteins is rather unlikely to be disputed. Nonetheless, it has it should be alternated with some others, for instance, immunological techniques and near-infrared spectroscopy. A great number of scholarly works have been dedicated to the study of species adulteration. The main reason for this avid attention is that this substitution occurs not only in seafood. Very often, various types of meat like pork or beef can be replaced by soya. Moreover, in some cases, it is necessary to determine the degree of adulteration and this is one of the most important tasks.

Having examined scholarly works, examining this question, it is possible for us to argue that they support the idea of such experiment. Various authors argue that gel electrophoresis is an efficient tool for the determining species substitution provided that this procedure is carried out appropriately (Gayo et al, 2006, Hall, 1997). In fact, one of the major advantages of this strategy is that it offers the best ratio of speed and accuracy. Yet, they also urge us not to forget that this is not the only method. The results of this study should be taken into consideration, provided that that further research is undertaken. The subsequent studies can evaluate the effectiveness of methods, determining species substitutions.

Therefore, at this moment we can conclude that the division of proteins according to their molecular weight is a helpful tool for deterring whether the biological material has been substituted or not. Such chromatographic technique as gel electrophoresis can be effectively utilized for this purpose. This research should be regarded only as a constituent part of larger fields of study, chromatography and food forensics.

References

Carpenter T. Moershel G. & Richard Dreger. Laboratory Manuel. University of Texas.

Dominges E. Perez, M. Puyol P, & Calvo M. “Use of immunological techniques for detecting species substitution in raw and smoked fish”. The Journal of Food Quality Control. 1997, pp 279-281.

Garret R. & Grisham C. Biochemistry. Cengage Learning, 2005

Gayo. J. Hale S. & Susan B. “Quantitative Analysis and Detection of Adulteration in Crab Meat Using Visible and Near-Infrared Spectroscopy” Journal of Agricultural and Food Chemistry. 2006 54 (4), pp 1130–1136.

Hall. G. Fish processing technology. Springer, 1997.

McClatchey. K. Clinical laboratory medicine. Lippincott Williams & Wilkins, 2002.

Scheppler J., Cassin P., & Gambier R. Biotechnology explorations: applying the fundamentals. ASM Press, 2000.

Stanley. J. Essentials of immunology & serology. Cengage Learning, 2002.

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