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Western Blotting which is Used to Isolate Myosin Light Chain - Research Paper Example

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The paper 'Western Blotting which is Used to Isolate Myosin Light Chain' presents protein blotting that is a potent as well as an important technique for the immunodetection of proteins subsequent to electrophoresis, especially proteins that are present in low quantities/ abundance…
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Western Blotting which is Used to Isolate Myosin Light Chain
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Protein blotting is a potent as well as important technique for the immunodetection of proteins subsequent to electrophoresis, especially proteins that are present low quantities/ abudance. Furthermore, it is frequently used in molecular as well as cell biology. The transfer of nucleic acids or proteins to nitrocellulose membranes is known as blotting. Moreover, blotting includes physical sample deposition as well as removal SDS-PAGE gels. In this technique, proteins separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE gels) are usually moved to an adsorbent membrane support under the effect of an electric current in a process known as western /protein blotting. What is more, western blotting assists scientists to isolate specific proteins e.g. Myosin light chains from a composite mixture of proteins extracted from cells. Besides, this technique is dependent on three elements to realize this chore: (a) isolation based molecular size, (b) transfer to nitrocellulose and (c) immunodetection to visualize. In this lab experiment, western blotting was used to isolate myosin light chain from composite muscle cell extracts of closely as well as distantly related species of fish. Introduction Western blot is a technique used in scientific investigations to isolate as well as identify polypeptides. In this method an assortment of polypeptides are resolved on the basis of molecular weight, and hence by type, by gel electrophoresis. Consequently the proteins are then relocated to a micro porous membrane generating a band for each protein. The micro porous membrane is then incubated with antibodies particular to the protein being studied. The unattached antibody is then washed off leaving only the antibody attached to the protein investigated. The bound antibodies are then visualized by processing the film. Since the antibodies only bind to the protein being studied, only a single band should be detectable. Besides, the thickness of the band relates to the quantity of protein present; hence running a standard can demonstrate the quantity of protein present in the cell extract (Mahmood and Ping Chang, 2012). In this research protein gel electrophoresis (SDS-PAGE) as well as western blotting was used to specifically identify a subunit of myosin light chain from the composite proteins that constitute the muscle tissues of diverse fish species. Methods Preparation of muscle protein extract In the experiment, the proteins in fish muscles were extracted through the lytic action of a lysis buffer used to break open the muscle cells. The extraction buffer contained the ionic agent sodium dodecyl sulfate (SDS) as well as a strong reducing agent DTT-dithiothreitol. Moreover, SDS efficiently coats all the proteins in the sample with a negative charge and DTT disrupts disulphide bridges that give proteins secondary, tertiary as well as quartenary structure. SDS and DTT are contained in the lysis buffer (Laemmli sample buffer). The proteins were heated further at 95 degrees centigrade for further denaturation. Once the extraction was complete, all the proteins in the sample were uniformly coated with SDS and carried equivalent negative charge density. Thereafter, SDS-PAGE electrophoresis, was used to separate polypeptides/ protein subunits based on molecular weight. Seperating proteins using SDS-PAGE At this stage the proteins in muscle cell extracts were separated according to their molecular weights via gel electrophoresis. Through the use of an electric current, proteins coated in SDS containing sample buffer are separated in a sieving gel matrix that seperates proteins according to their size. Precision plus protein kaleidoscope pre-stained protein standards were used to visualize the proteins in the muscle extracts. Furthermore, these genetically engineered proteins have dyes covalently bound to them and resolve into multi-colored bands that moved down the gel during electrophoresis. Additionally, the blue tracking dye (bromophenol blue) in the sample buffer was also used to monitor the progress. The blue dye is negatively charged and is smaller than most known proteins, so it moved towards the positive electrode marginally ahead of the proteins. Western blotting After the muscle extract proteins were separated by SDS-PAGE they were transferred out of the gel and blotted onto a membrane to facilitate antibody detection of myosin i.e. antigen. Firstly, sandwich was carefully prepared from the SDS gel and the nitrocellulose membrane. Secondly, an electric current was then passed through it, and the proteins were ultimately blotted onto a nitrocellulose membrane in a mirror image configuration to that found in the gel. Nitrocellulose membrane serves as a solid support for proteins bound to its positively charged surface. Immunodetection of Myosin light chains Because antibodies bind as well as identify specific proteins, they were utilized in this laboratory experiment as the perfect tool for identification of the antigen i.e. Myosin light chains (MLC1as well as MLC2) from an assortment proteins immobilized on the membrane. During the immunodetection the primary antibody was first added to the blot followed by the secondary antibody conjugated to HRP (an enzyme that catalyzes the oxidation of colorimetric substrate so that myosin light chains i.e. protein of interest; can be identified) and lastly, the colorimetric (color generating) enzyme substrate is added to the membrane and incubated to allow color development. Result Following immunodetection, purple bands developed on membrane exactly where myosin protein bands were located. Discussion The colorimetric substrate in the kit used for this experiment was 4CN (4-Chloro-1-Naphthanol). When oxidized by HRP in the presence of hydrogen peroxide, this colorless solution formed a purple/gray precipitate that binds to the membrane at the antigen i.e. Myosin light chains location. Moreover, HRP color detection reagent is light sensitive and ought to be kept in the dark at all times. Sources of error Although the process for western blotting is straight forward, many errors can arise, resulting unanticipated results. These problems can be categorized as follows: (a) no bands, (b) unexpected bands (c) faint bands (d) high background on the blot as well as (e) uneven spots on the blot (Mahmood and Ping Chang, 2012). Trouble shooting No bands can occur due to a number of reasons associated to antibody, antigen, or buffer used. If an incorrect antibody is used, i.e. primary or secondary, the band will not appear. Furthermore, the concentration of the antibody must also be suitable; if the concentration is too low, the signal may not be visible. It is imperative to recollect that some antibodies not applicable for western blot. Additionally, no bands can occur due to lowest concentration or lack of the antigen. In this case, another antigen source can be used to check whether the error is caused by sample or the antibody. Likewise, protracted washing can also diminish the signal. Additionally, buffers can also add to the problem. It should be made certain that buffers like the transfer buffer and running buffer not contaminated (Mahmood and Ping Chang, 2012). On the other hand, unexpected bands can be caused by protease degradation, which generates bands at unanticipated locations. In this case it is logical to use a new sample (antigen) or change the antibody. If the protein seems to be in too high of a location, then reheating the sample can help to disrupt the quaternary (3D) polypeptide structure. Likewise, blurry bands are usually a consequence of air bubbles or high voltage present during transfer. It must be guaranteed that the gel is run at the optimum voltage, and that the transfer sandwich is prepared appropriately. Furthermore, replacing the running buffer can also help resolve the problem. Non-flat bands can also be the consequence of fast travel through the gel, because low resistance. To solve this, the gel must be optimized to fit the sample. In conclusion, white (negative) bands on the film are attributable to too much antibody or protein. High background is usually the result of high concentration of the antibody, which can bind to nitrocellulose membranes. Another problem might be the buffers, which may be exhausted. Protracted washing time can also assist in reducing the background (Mahmood and Ping Chang, 2012). . Conclusion Following this laboratory experiment, it can be concluded that western blotting technique proved to be a significant as well as a potent tool in identification of Myosin long chain proteins from the composite proteins that constitute the muscle tissues of diverse fish species. References Mahmood, Tahrin, and Ping-Chang Yang. “Western Blot: Technique, Theory, and Trouble Shooting.” North American Journal of Medical Sciences 4.9 (2012): 429–434. PMC. Web. 1 Dec. 2014.  Biji T. Kurien, R. Hal Scofield. “Introduction to Protein Blotting.” Methods in Molecular Biology Volume 536 (2009): pp 9-22. Date: 25 Feb 2009. Read More
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