SDS Lab 03.01

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Miller-Motte Technical College, Cary *

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Feb 20, 2024

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1 EXERCISE 5: Separation and detection of proteins I. OBJECTIVES A. To separate red blood cell cytoskeletal proteins on an SDS-PAGE gel, B. To visualize these proteins with Coomassie Brilliant Blue staining, C. To transfer these proteins to a nitrocellulose membrane for subsequent immunoblot detection (Western blot), D. To probe for albumin by immunoblot (Western blot). II. BACKGROUND: ENRICHING FOR AND DETECTING THE PROTEIN OF INTEREST A. Introduction The thousands of different proteins found in cells play important roles in most cellular functions including metabolism, interactions between cells, and structural support. As scientists, we often wish to study specific proteins to understand their functions within the cell. Tools for identifying and visualizing proteins prove invaluable to these efforts. The laboratory exercises that you complete over the next two weeks will help you explore some of the possibilities and limitations of working with proteins. Parts B and C (described below) have been completed for you due to time constraints. To understand the parts of this exercise that you complete, however, you need to understand what we did on your behalf. B. Isolating Red Blood Cells For this lab, we started with sheep’s blood. Whole blood consists of red blood cells, white blood cells of various kinds, platelets, and plasma. All of these components have large protein pools, but we are only interested in red blood cell cytoskeletal proteins. The first step required for preparing this lab, then, was to isolate red blood cells. The different densities of the various blood components provided a mechanism for doing so. In the presence of an anti-coagulant, tubes containing whole blood were

2 spun rapidly in a centrifuge. Centrifugation exerts strong forces on samples placed in the centrifuge. The sheep’s blood was exposed to forces on the order of 15,000 times the force of gravity for about 30 minutes. This separated the whole blood into layers based on the densities of the various blood components. This process is called differential centrifugation. The blood layer with the greatest density contained the red blood cells. To isolate red blood cells, we discarded the upper layers and washed the red blood cells several times. Because of its abundance, a 68 kD (kiloDalton) plasma protein called albumin likely contaminates the preparation. We will measure this contamination during the second week of this exercise. C. Isolating Membrane Proteins The proteins we will examine are the cytoskeletal proteins. These give the cells their shape and play roles in transporting material within cells. Recognizing that all of the cytoskeletal proteins are linked directly or indirectly to the plasma membrane, we purified our protein pool by isolating just the plasma membrane ‘fraction’ of the cell. The process we used, called fractionation, began with lysing (i.e. breaking open) the cells. Cells can be lysed using various methods including detergents, mechanical disruption, and osmotic shock. To remove the cell contents without breaking up individual organelles or the plasma membrane itself, we used the relatively mild osmotic shock method. Placing the cells in a hypotonic buffer caused the cells to swell until the membranes developed holes. (You should be able to explain why this happened. Review osmosis in your textbook if this is unclear.) The holes in the plasma membrane allowed the cellular contents to flow out, without removing plasma membrane associated proteins or lipids. Cells prepared in this way are referred to as “ghosts” because they retain a cellular shape but are empty. Isolating the ghosts away from the organelles involved differential centrifugation. The washed red blood cell ghosts comprise the sample that you will work with in lab.

3 D. Size Separation of Proteins SDS-PAGE (S odium d odecyl s ulfate p olya crylamide g el e lectrophoresis) is a technique which allows us to separate proteins based on molecular size. The initial difficulty lies in the fact that proteins are composed of different amino acids. Each amino acid has a characteristic three-dimensional shape and charge (negative, positive or neutral). Linking these amino acids together covalently in a polypeptide gives each protein a characteristic three-dimensional shape and charge. This raises the question of how SDS-PAGE uses an electric current to separate proteins only on the basis of size. The answer involves the methods used to prepare the samples for SDS- PAGE. Sodium dodecyl sulfate (SDS) is a strong anionic detergent that destroys all secondary and tertiary protein structure by disrupting non-covalent interactions (hydrophobic interactions, ionic bonds, and van der Waals forces) within a protein. Second, the protein samples are heated to 100° C which disrupts hydrogen bonding. Finally, a reducing agent (dithiothreitol in our case) is necessary to break disulfide bridges within and between polypeptide chains that help maintain the protein folds. Preparing samples in this way linearizes polypeptide chains. SDS has an additional essential role of coating the linearized polypeptide with a uniform negative charge, overwhelming the charge differences due to the amino acid charges. The size separation of proteins (or any other macromolecule) is accomplished by resolving them through a semi-solid matrix, which in our case is made of polyacrylamide. Migration of the negatively charged proteins through the polyacrylamide gel is accomplished by applying an electrical current. The denatured proteins are introduced to the gel at the cathode (negative pole) and as they are negatively charged due to the SDS, they will migrate towards the anode (positive pole). Smaller molecular weight proteins migrate quickly toward the anode, while larger proteins are retained near the top of the gel. The study of red blood cell membrane and cytoskeletal proteins will utilize 12% acrylamide gels. These gels resolve proteins in the molecular weight range between 15 and

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4 80 kD. The gels you will use in class have been prepared for you. You will load your protein samples into the wells of the gel. After the gel is run, you will be looking in each lane (the long strip under the well) for the bands of protein. In order to determine the actual size of the proteins, we will be including a molecular weight marker, also called a size standard. The marker is a preparation of proteins of known sizes. After the gel is run, these proteins can be used as a ruler to determine the sizes of proteins from the experimental sample. For instance, a protein that resolves halfway between the 25 kD marker and the 32 kD marker is going to be ~ 29kD. Smaller (i.e. shorter) proteins migrate (or “run”) faster through the gel because the gel impedes their progress less than larger (i.e. longer) proteins. This means that the smallest marker will be farther down the gel and the largest will closer to the top. When analyzing your gel, notice that the markers are not equally spaced. Bands separate less near the top. Also be aware that sometime small proteins run off the bottom of the gel or are masked by the blue dye front. The proteins that you resolve by SDS-PAGE will be visualized using two different techniques. Coomassie Brilliant Blue binds to all polypeptides and will give the otherwise colorless proteins a blue color. This method of detecting proteins does not work for low abundance proteins. As described in the following section, you will also use a far more sensitive method for detecting specific proteins that involves antibodies. E. Immunodetection of Proteins A technique that is commonly used to identify the presence of a given protein within a preparation is called electroimmunoblot analysis (or Western blot analysis). Following SDS-PAGE, you will electrophoretically transfer the proteins to a nitrocellulose membrane. This membrane, or ‘blot’, provides a solid support for the proteins, whereas the acrylamide gel is both very delicate and prevents access to the proteins within. After transferring the proteins to the membrane, you will incubate it with an antibody that recognizes albumin. Antibodies are extremely specific for proteins and are produced by B-

5 Iymphocytes in the body in response to foreign proteins. Antibody binding to foreign proteins (antigens) targets the antigens for removal from the body. An antibody raised against a specific antigen is called the primary antibody . Albumin is the antigen for the primary antibody you will use. Primary antibodies have a variable region and a constant region . The variable region is what makes a particular antibody specific to a particular antigen. The constant region, on the other hand, is exactly the same for every antibody produced within an animal. Every antibody made in a rabbit, no matter what the variable region recognizes, will have an identical constant region. A primary antibody against albumin that is mixed in with our blots will bind exclusively to albumin through its variable region; the constant region will ‘stick out’ from the blot. Primary antibodies can be used alone (direct immunodetection), but generally another step using secondary antibodies is included. A secondary antibody is made by using the constant region of the primary antibody as the antigen for the secondary antibody. Use of the secondary antibody allows us to amplify the signal dramatically. For example, if 10 primary antibodies were able to stick to each strand of albumin, we would have 10-fold amplification of the signal. If 10 secondary antibodies stuck to each primary, we would have 100-fold amplification. Finally, we need some method to visualize this protein-antibody complex. The secondary antibody can be detected by one of several methods. The secondary can be tagged radioactively, fluorescently, with a gold bead (for electron microscopy), enzymatically. Enzyme tags (e.g. alkaline phosphatase or horseradish peroxidase) convert a colorless solution to a colored precipitate. Antigen detection using a labeled secondary antibody is termed indirect immunodetection. After washing off excess anti-albumin antibodies (primary antibody), you will add a secondary antibody (against the constant portion of the anti-albumin antibody) that is complexed with horseradish peroxidase. This enzyme, in the presence of the chemical H 2 0 2 (hydrogen peroxide) and the chromophore 4-chloro-napthol, forms a purple precipitate identifying the presence of albumin on the nitrocellulose membrane.

6 III. PROCEDURES This experiment will span 2 weeks . Week one will include sample preparation, running of the SDS-PAGE, and immunoblotting. Week two will include a library research presentation, immunoblot development, and gel and blot analysis. WEEK ONE A. Prepare the samples, load gel 1. Turn on the hot plate at the back table in the laboratory and heat a beaker of water to boiling. 2. Prepare your UNKNOWN SAMPLE : Place 200 μL of erythrocyte ghosts in a microcentrifuge tube. Add 200 μL of 2x sample buffer into the same microcentrifuge tube. Add 10 μL of 1M dithiothreitol (DTT) into the tube. Mix the contents by GENTLY pipetting up and down. 3. Prepare your POSITIVE CONTROL : A. First, prepare 0.1M DTT stock solution by adding 10 μL of 1M DTT and 90 μL of distilled water in a clean microcentrifuge tube. B. Into a fresh microcentrifuge tube, place 30 μL of mouse plasma. Add to the microcentrifuge tube 40 μL of 2x sample buffer and 10 μL of the 0.1M DTT (prepared in step A above) and mix by GENTLY pipetting up and down. 4. Place the sample and control in a floater. Place in boiling H 2 O for 5 minutes. 5. While samples denature, remove comb from gel (slide gently but firmly upwards). Flush out the wells using a 200 μL pipette. Ensure that the SDS Running buffer comes to the top of the gel plates (interior) and halfway up the plates (exterior). Group 2

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7 6. After heating samples, load gels as follows (skip even numbered lanes): Lane 1: 10 μL of pre-stained weight marker Lane 3: 10 μL of sample Lane 5: 15 μL of sample Lane 7: 20 μL of sample Lane 9: 20 μL of positive control 7. Place lid on gel box. Make sure you connect the gel box to the power supply correctly. Run at 200 V for approximately 33 minutes – until the blue dye front gets to the very bottom of the gel plate. 8. When run is complete, remove lid from box. Gently lift out inner chamber, and pour running buffer into sink. Open clear plastic “gates” to release the gel plates. Open the gel plates CAREFULLY – acrylamide gels tear easily. TEAM A : You will stain your gel using Coomassie Brilliant Blue. WEAR GLOVES or your hand will turn blue. Pour some dye into the labeled plastic container. Wet the tips of your gloves with Coomassie dye, so that the gel will not stick to your gloves. Gently ease the gel into the container. Ensure that the gel is flat and not folded. Seal the container and dispose of the gloves. The gels will be destained for you. TEAM B: You will perform electroimmunoblotting on your gel. Please be sure that your Team B members watch as this procedure is performed.

8 B. Electroimmunoblotting 1. Pour transfer buffer into preparation container. Wet the transfer sponges and Whattman paper. Assemble the gel sandwich in the preparation container as illustrated below. (Note: Gloves must be worn when handling nitrocellulose.) With the black side of the holder on the countertop, be sure that the nitrocellulose is on top of the gel. 2. Using the piece of pipette, gently roll bubbles out of the sandwich, ensuring a complete connection between gel and nitrocellulose. Put 2 more wet sponges on top of the sandwich. Close sandwich holder and leave in transfer buffer until apparatus is assembled. 3. Put red/black transfer unit in transfer box. Put in white ‘cooling unit’ (white container with ice in it). Slide gel sandwich holder into the transfer unit. Be sure that the black side of the holder faces the black side of the transfer unit. Pour transfer buffer from tray into the gel box. Continue to add fresh transfer buffer until the buffer comes up to the ‘flare’ (1/2 inch from top). Sponge (1) Wet Whattman paper (2) Nitrocellulose paper (1) Wet Whattman paper (2) Gel (1) Black side of ‘sandwich holder’ Sponge (1)

9 6. Place the lid on and run for 1 hour at 300 mA. Heating the transfer buffer changes its conductance. If the power supply reaches the limit (a loud buzzing noise), turn down the amperage until buzzing stops. 7. After the run, remove the entire contents of the transfer box and disassemble the sandwich. The nitrocellulose blot is to be placed in prepared blotting/antibody solution. Gel and paper are to be discarded. The sponges, holder, and transfer box are to be rinsed thoroughly. WEEK TWO Teams A and B will work together. Begin the final stages of immunoblotting. Then examine the destained gel from last week. The following table lists the sizes of the molecular weight markers that you loaded on the gel. Protein standard Size (kD) Myosin 200 Phosphorylase B 96 Bovine albumin 68 Ovalbumin 43 Carbonic Anhydrase 30 b-Lactoglobin 18 Lysozyme 14 C. Immunoblot detection (Western blotting) 1. Last week, blots were placed in blotting solution with a 1:1000 dilution of primary antibody (rabbit anti-albumin). 2. WEAR GLOVES AT ALL TIMES. Wash blot in 15 mL of washing solution for 2 minutes on shaker. Repeat 2 times. 3. Add 5 mL of blotting buffer to membrane. Tilt box slightly, creating a pool of solution in the corner of box. Using a micropipetter, add 10 μL of secondary antibody (goat anti- rabbit) to blotting buffer and NOT directly on the membrane. 4. Swirl to mix, and put on shaker for 1 hour.

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10 5. Wash 3 times as above (2 min/wash). 6. After the 3rd wash is discarded, place 5 mL of blotting buffer in tray containing the immunoblot. 7. Prepare the developer. Do NOT prepare the developer until you need it. Place 25 mL of blotting buffer in a beaker and add to it 5 mL of the 4-chloro- napthol reagent found in the freezer. Mix these reagents by swirling the beaker. Add 15 μL of cold hydrogen peroxide (stored in the refrigerator) to the beaker and swirl again. 8. Drain blot and add developer. Swirl gently until a strong color develops (this should be very quick). When color is established, rinse thoroughly in dH 2 O, and air dry on a paper towel. IV. CLEAN UP 1. All components of the gel boxes must be rinsed with water and left to air dry. 2. Discard all waste such as tips and gloves in the trash. 3. All solutions stored on ice or in the refrigerator must be returned to the cold storage. 4. Rinse out all plastic containers. Type text here

11 ANSWER SHEET FOR EXERCISE 5: Separation and detection of proteins Answering these questions will help you prepare to write the lab report. 1. What was the purpose of washing the red blood cells with saline prior to lysis? 2. What property of the lysis buffer caused the red blood cells to lyse? 3. What are the differences between the primary and secondary antibodies used in this study? 4. What are the advantages of using indirect immunodetection over direct immunodetection? 5. Antibodies are made when animals detect a foreign antigen. All mammals carry albumin in their blood. How was a rabbit antibody against albumin created? Was this lethal to the rabbit? Explain you reasoning. The purpose of washing the RBC prior to lysis is to get rid of any contaminates like albumin. This will allow us to have a more pure sample and the contamination was meaured in week 2. By placing the cells in the hypotonic buffer, it allows them to get larger until the membrane developes holes in which the cellular components will exit the cell without disturbing the plasma membrane The primary antibody binds to the specific antigen and the secondary antibody uses the c onstant region of the primary antibody as the antigen. The purpose of using the secondary a ntibody allows the constant region to be amplified when we perform the blot. Direct immunodetection only uses the primary antibody meanwhile indirect immunodetection uses both primary and secondary antibodies which allows the benefit of an amplified signal. The antibody in the rabbit was created by isolating albumin from a sheep and injecting it into the rabbit. The rabbit then generates antibodies for the antigen and is isolated from i ts blood. It may or may not be lethal to the rabbit depending on how much blood is drawn.

12 6. Albumin is not a cytoskeletal protein. What was the purpose of using antibodies to detect albumin in our cytoskeletal preparations? 7. Why was it necessary to transfer the proteins separated by SDS-PAGE onto nitrocellulose membranes prior to incubation with anti-albumin antibodies? 8. Why did we electrophoretically transfer the proteins resolved by SDS-PAGE onto nitrocellulose? Can you suggest another way to transfer proteins from a SDS gel to a membrane? 9. Why did we wash the nitrocellulose blots with blotting buffer three times after incubating with the primary and secondary antibodies? Albumin is a plasma protein and we used the antibodies to detect albumin in the cytoskeletal preparations because plasma proteins, like albumin can still be present in the sample. The nitrocellulose membranes provide a membrane like function for the proteins to be transferred to the membrane to then analyze Trough electrophorectically transferring the proteins will allow us to end up with an effective transfer of proteins. Two other methods is semi-dry blotting and dry blotting, these two methods require minimal cleanup and less time. We washed the nitrocellulose blots three times to remove any remaining primary antibody. If we didn’t wash it three times, then we could have generated unexpected results and/or an unwanted reaction.

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SDS Lab 03.01

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