Cell Bio Essay

Western blotting is an analytic technique used around the world for many reasons; detecting infections, diseases, and particular proteins in a tissue - Cell Bio Essay introduction. When western blotting, first the proteins are extracted and undergo Gel Electrophoresis, next it goes through elecroblotting, and finally detects the proteins. When the results after every step is completed, the membrane is analyzed based on where the bands are present in the gel. It has been told that actin and myosin is present in fish, making it tougher to chew in most cases. To test the theory of actin and myosin being present in fish making it tougher to chew was the purpose of this experiment. The proteins from Rainbow trout, Catfish, Pollock, Salmon, Tilapia, Mahi-mahi, Cod, Lake Erie Perch, KD and Actin/Myosin standard were analyzed in the study and ended up coordinating the findings in the study with the findings in the theory. We discovered that both Actin and Myosin were present in all types of fish and that some fish had more bands than others, thus having more variations. Therefore, proving that our theory was certainly correct about the actin and myosin making the fish tougher to chew. Introduction

The western blot, also known as the protein blot or immunoblot, is a method used to separate proteins by molecular weight. Western blotting is used to detect proteins in a tissue or extract. In a previous experiment a gentleman by the name of K. Nockler and many others were studying an infectious food borne disease called Human Trinchinellosis. Human Trinchinellosis is a disease caused by ingestion of infected pork, meat and other foods in which animals can be exposed to this parasite. He analyzed sera from pigs using a western blot, for the detection of anti-Trichinella-IgG. The results from his experiment using western blotting was that 144 of the sera from pigs were Trichinella-free and 159 pig sera were infected. His study and our experiments are similar, but instead of trying to find a disease or infection in this study, actin and myosin was the focus. The purpose of this study was to examine if actin and myosin were present in different types of fish proteins by observing the molecular weights of the proteins using kaleidoscope, by western blotting. We examined Rainbow Trout, Catfish, Pollock, Salmon, Tilapia, Mahi-mahi, Cod, Lake Erie Perch, KD and Actin/Myosin Standard proteins and analyzed where the bands of the proteins were present on the membrane and compared them to where the actin and myosin bands fell in the membrane. Method

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A western blot experiment is broken down into four different stages. The first stage is to extract the proteins. In order to do this you start by taking six 1.5 microcentrifuge tubes and six screw cap tubes and label with each of the different meat samples. Then, one inserts 50 μl of Laemmli sample buffer into to each microcentrifuge tube. A piece of each meat sample is then cut and transferred to its labeled microcentrifuge tube. The tube must be flicked 15 times and incubated at room temperature for 5 minutes to agitate the proteins. The 15 μl buffers from each centrifuge is transferred into its labeled screwcap microcentrifuge tube using a micropipette and then heated to 95°C to extract the protein. The second stage is to use the buffer to undergo Gel Electrophoresis. To do this first, prepare the Mini-PROTEAN TGX gel. When you have the gel clamped into the electrode assembly you then place it into the gel tank. The inner chamber is filled with 1XTGS. Next step is to load the gel with the fish samples, actin and myosin standard, and the kaleidoscope. After loading the gel, the lid is placed on the electrophoresis chamber and it is ran at 200 V for 30 minutes. Third stage consists of the electroblotting of the western blot. In this stage you prepare the gel to be transferred into a membrane. When the gel is finished running, remove and equilibrate in blotting buffer for 15 minutes on a rocking platform in a gel staining tray.

After, blotting sandwich is made composing of two fiber pads soaked in buffer, the gel made from the previous stage, and two membranes, roll out any air bubbles. The gel holder clamped the sandwich together and then inserts into the Mini Trans-Blot inner module. Then the chamber is filled with blotting buffer and is to run at 20V for two and a half hours. The third and final stage of a western blot is detecting the proteins. After the electrophoresis is completed, the blocking solution is discarded and the membrane is incubated with a 10ml primary antibody for ten minutes on the oscillator. The membrane is then rinsed with 50ml of wash buffer and then discarded. It was then added a second time, put on the oscillator for three minutes, and discarded again. Then incubate the membrane with a secondary antibody and put on the oscillator for ten minutes. After incubating apply wash buffer and discard it and then apply it again, oscillating it for three minutes this time around. Then in order to develop color within the bands a coloring detecting reagent is added for ten to thirty minutes on an oscillator. Finally the membrane is rinsed with distilled water and blot dry with a paper towel then air dry for thirty minutes. Results

Cod contained the most number of bands, showing the presence of more variation of proteins in comparison to the other fish. Cod and Pollock had similar markings. Rainbow trout and Tilapia contained the least number of bands and were hardly seen, showing that it had fewer proteins than the other types of fish.

Lane 1| 15μl| KD|
Lane 2| 15μl| Actin/Myosin Standard|
Lane 3| 15μl| Lake Erie Perch|
Lane 4| 15μl| Cod|
Lane 5| 15μl| Tilapia|
Lane 6| 15μl| Rainbow Trout|
Lane 7| 15μl| Pollock|
Lane 8| 15μl| Mahi-Mahi|
Lane 9| 15μl| Salmon|
Lane 10| 15μl| Catfish|

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