Western Blotting

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Western Blotting is used to Identify a Subunit of Myosin
Light Chain in the Proteins of All the Different Fish.
Chae Yee
Kent State University:
BSCI 30140 Lab

Abstract
Western Blotting can be used to detect the Myosin actin light chain in different species of fish and is used to distinguish from different species based on variation, commonality, or evolutionary divergence. First, proteins are extracted from the tissue and loaded into a gel matrix. The matrix will separate the proteins according to size using an electric current. Proteins that are separated after are blotted from the gel and onto a paper membrane. An antibody is then added to the membrane paper and causes a colored reaction. Following the reaction, the results help detect and quantify a single protein among hundreds of other proteins in the sample. Western blotting is used during this procedure to test that proteins can be indicators of genetic and evolutionary similarity. Results show that the different species of fish contain the myosin light chains that are equivalent in molecular mass, which then means they are similar in their evolutionary relationships.

Western Blotting is used to Identify a Subunit of Myosin Light Chain in the Proteins of All the Different Fish. Introduction
Western blotting is a technique in biological research that allows scientists to identify and quantify specific proteins among a protein mixture. The method that is used is a “protein mixture is applied to gel-electrophoresis in a carrier matrix (SDS-PAGE) to separate a protein by size and charge. Next, the separated protein-bands are transferred into a carrier membrane. The proteins are then accessible for anti-bonding in order to detect them” (Antibodies-online.com, 2012). Myosin is a muscle protein that is essential to animals for survival and has remained stable over time. The myosin light chain can be compared from different species for evolutionary divergence and similarities. The objective of this study is to test that proteins can be markers to help relate genetics and evolution within a species of fish. Western blotting is used to identify a myosin light chain from the proteins containing muscle tissues of different kinds of fish and tests that proteins are indicators of genetic and evolutionary likeness (Department of Biological Sciences, 2013).

Method
The lab will begin with preparing muscle protein extracts by removing proteins from the muscle tissues of different fish. First, muscle tissues from different species of fish are added to five 1.5 ml fliptop micro tubes that are labeled. Laemmli sample buffer is then added to each tube. After, the micro tubes were flicked for a numerous amount of time to agitate the tissue in the sample buffer. The next step was put the tube in the incubator for five minutes at room temperature. Once it cooled down the buffer was extracted from the tube, leaving the fish sample still in there. The fish sample was then heated at 95°C for five minutes.

The second part of the lab is to separate the proteins using SDS-PAGE. The first step is to set up the apparatus that will be used in the experiment; which this is the polyacrylamide gel electrophoresis. A Ready Gel cassette is prepared and is placed into the electrode assembly. The electrode assembly is then placed in the inner chamber of the tank and filled with TGS gel running buffer. Once this is complete, heat the fish samples and actin and myosin standards for 2-5 minutes at 95°C and then load the gel. Run the electrophorese for about 30 minutes at 200 V. When the time is up remove the gel from the cassette and transfer it into a container with 25 ml Coomassie stain and let it stain for one hour with gentle shaking.

The previous two steps were extracting the proteins and separating them by their size. The remaining of the lab deals with the usage of antibodies to help identify the myosin light chain in the tissue. First, chop off the bottom and top of the gel and...
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