Ex3_4_ComparativeProteomics_SP24

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Texas A&M International University *

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3012

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Biology

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Feb 20, 2024

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BIOL3012 Cell Biology Laboratory S24 Exercises 3 & 4 Comparative Proteomics Muscle Proteins and Myosin through SDS-PAGE and Western Blotting Schedule Guide: Week 5 Feb 20 FYI: no lab quiz on Feb 20! No lab sheet is due. You will perform Ex 3, SDS-PAGE: Muscle Proteins Separation and Part of Exercise 4: Western Blotting (Feb 20 will cover Lessons 2 and 3 from the protocol hand-out. But please read Lesson 1. Week 6 Feb 27 1) Lab Quiz at the beginning of lab: post coverage = SDS-PAGE and Western Blot from Feb 20 while pre-coverage = lesson 4 page 8 from protocol hand-out; 2) continuation of Exercise 4: Western Blotting: Probing Myosin; 3) Worksheet Due at the end of lab period. Background: Proteomics is a field of study that focuses on proteins, their structures, and their functions. The proteome, or the complete set of proteins expressed in a given cell or organism, can vary depending on factors such as cell type, life cycle stage, and environmental conditions. In this series of laboratory exercises, you will have the opportunity to explore two tools commonly used in protein research. First, you will examine myosin in fish muscles using Western blotting. Secondly, you will observe fish muscle proteins through visualization in a polyacrylamide gel using SDS-PAGE . Western blotting is a method that involves using antibodies to detect specific proteins based on unique structural elements, or antigens, in a given sample. Gel electrophoresis separates proteins by size or mass, and the SDS-PAGE technique is specifically designed to separate proteins according to size. The proteins are transferred to a membrane for Western blotting. Since you are trying to separate many different protein molecules of different shapes and sizes, you first want to denature the proteins. The proteins no longer have any secondary, tertiary or quaternary structure (i.e. you want them to retain only their primary amino acid structure). You use SDS to denature all proteins to the same linear shape (Figure 1). Figure 1. Schematic diagram of protein denaturation using SDS. Refer to this hand-out to prepare you for the pre-coverage part of lab quiz 3, Feb 13.

If the proteins are denatured and put into an electric field, they will all move towards the positive pole at the same rate, with no separation by size. Therefore, you need to put the proteins into an environment allowing different-sized proteins to move at different rates. The environment of choice is polyacrylamide, a poly mer of acrylamide monomers. When this polymer is formed, it turns into a gel, and the use of electricity will pull the proteins through the gel, so the entire process is called polyacrylamide gel electrophoresis (PAGE). A polyacrylamide gel is not solid but is made of a labyrinth of tunnels through a meshwork (Figure 2). Figure 3 shows a cartoon gel. Figure 2. A polyacrylamide gel is not solid but is made of a labyrinth of tunnels through a meshwork. . Figure 3. Top view of an SDS PAGE after the current has been on for a while (positive pole at the bottom) and then turned off. The molecular weight standards (lane 1) are used to measure the relative sizes of the unknown proteins. You stain the proteins and see how far they moved through the gel (until you stain them, they are colorless and thus invisible). On the other hand, if the gel will be used for Western blotting you do not stain gels. Remember that SDS-PAGE separates proteins based on their primary structure or size but not amino acid sequence . Therefore, if you had many copies of two different proteins that were either 500 amino acids long or approximately the same mass, they would travel together through the gel in a mixed band. As a result, you would not be able to use SDS-PAGE to separate these two proteins of the same molecular weight from each other. Western blotting is an invaluable tool in cell and molecular biology. By performing a Western blot, one can determine the presence or absence of a specific protein and measure the amount of protein produced by a cell or tissue. To execute a Western blot, the proteins that have been separated in an SDS-PAGE gel must be transferred to a nitrocellulose membrane. This transfer can be achieved through capillary action or electrophoretic mobilization. Following the transfer, the proteins should adhere to the paper in the same position as in the gel. The negatively charged proteins are attracted to the positively charged nitrocellulose membrane. To prevent the non-specific binding of antibodies, a blocking step is carried out using a protein solution that will not react with antibodies. Common blocking agents include powdered

non-fat milk, gelatin, or bovine serum albumin. The membrane is then washed with solutions containing primary and secondary antibodies. The primary antibody is specific to the protein of interest, while the secondary antibody is specific to the immunoglobulin protein of the primary antibody. Secondary antibodies are conjugated to markers that enable detection, such as an enzyme that creates light or a colored product that can be detected and recorded. The objectives of the lab exercises are to (1) set up and run SDS-PAGE, (2) set up and run Western blotting, (3) acquire the necessary technical knowledge to perform electrophoresis and Western blotting, and (4) detect the presence of myosin in the given samples. Materials:  fish samples  Protein molecular mass standard  Polyacrylamide gel  Electrophoresis chamber and buffer  Western blotting kit (nitrocellulose transfer membrane) Reagents:  Distilled Water  Blocking solution  Wash buffers  Primary and secondary antibodies  Secondary detection reagent Please be advised that the methodology for the upcoming lab exercises is available on BlackBoard in a separate file, labeled Ex3-4 methodology hand-out. It is highly recommended that you read the methodology hand-out, including the callout information prior to the scheduled lab exercises: (1) February 15/16- Lessons 1-3 and (2) February 22/23- Lesson 4. It is essential that you familiarize yourself with the protocols outlined in the methodology hand-out to ensure a clear understanding of the lab exercises. Failure to do so may impact your performance and efficiency. If you have any questions or concerns, please do not hesitate to consult with your lab professor/instructor. Use the figure below to show how to set up the “Gel-nitrocellulose Membrane Sandwich” -At the end of the lab period you should be able to complete the drawing. It is essential to correctly assemble the "Gel-nitrocellulose membrane sandwich" to transfer proteins effectively. _ _ PAD PAD +

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