Western Blotting Lab 4 Guide F23 PDF

Western Blotting (Blot prepared by BIOL 2020 students) Last lab you electrophoresed your stressed and non-stressed cell lysate proteins through an acrylamide gel and transferred them onto nitrocellulose membrane. Today you will use those membranes to perform a western blot to identify a specific pr...

Western Blotting (Blot prepared by BIOL 2020 students) Last lab you electrophoresed your stressed and non-stressed cell lysate proteins through an acrylamide gel and transferred them onto nitrocellulose membrane. Today you will use those membranes to perform a western blot to identify a specific protein of interest in your cell lysates! This common technique uses antibodies to assess protein expression. In our case, it can help determine whether the experimental conditions alter the expression (i.e. up or down-regulation) of a particular protein. Today you will probe for either Hsp27 or p53. These proteins are involved in cell stress and apoptotic signaling pathways. You will also probe for tubulin, as a procedural check. Learning Outcomes: By the time this lab is complete, you should be able to: • • Describe western blotting theory. Interpret western blotting data. BEFORE Lab 4, you must: • Carefully read the rest of this document and print it. • Complete the Lab 4 flowchart. Complete the pre-lab quiz on Brightspace. (Heads-up, the quiz includes questions from your previous labs!) • Assessments Related to the Lab: • Complete the In-Lab Worksheet (provided to you during the lab) and submit before you leave. McCarville, Garant and Tatar (2023/2024) Antibodies: Antibodies are proteins produced by an animal’s immune system. Because of their high degree of specificity against a target protein (called an antigen), they are powerful tools in cell biology research. Antibodies against a given protein can be raised in and collected from an animal host, often a mouse, rabbit, or goat. Such antibodies typically belong to the IgG class and have a Y-shaped structure. The base of the Y is the constant domain, which is identical for all IgG-class antibodies from a single species. The branches of the Y are the variable regions, which complement the shape of the antigen and allow binding to occur. There are two general approaches to generating antibodies for use in research: Polyclonal Antibodies: Traditionally, antibodies are made by injecting an animal host (often a mouse, rabbit, or goat) with the purified antigen, or protein of interest. The animal’s immune system will produce a variety of antibodies in response, and then those antibodies are harvested from the animal’s blood. This results in a mixture of antibodies, each specific for a different epitope on the protein of interest. These are referred to as polyclonal antibodies. Monoclonal Antibodies: In contrast, monoclonal antibodies are specific for the same epitope on an antigen. They are produced by injecting an animal host with an antigen, which triggers an immune response. Instead of collecting the antibodies from the animal’s blood, we remove some of the antibody-producing white blood cells. These cells are fused with immortal cells to produce hybridomas that will continuously divide in culture. A single hybridoma can be isolated and cultured to produce many copies of the exact same antibody. The advantages of monoclonal antibodies are that they are highly specific and less likely to randomly bind other proteins. Reporter Molecules: In molecular research, we need to visualize the binding of an antibody to its antigen. We typically purchase antibodies from biotechnology companies, and these companies will attach (i.e. conjugate) a reporter molecule to the antibodies. The reporter molecule might fluoresce under a microscope, or it could be an enzyme that would catalyse a reaction to produce a colour change or burst of light. Indirect Visualization: In research laboratories, it is common to make use of two antibodies, referred to as the “primary” and the “secondary”. The primary antibody recognizes the protein of interest and is named by stating its constant region identity, followed by its variable region specificity. For example, if you had a primary antibody that was produced in a rat, and recognized a protein named “kinesin-2”, the antibody would be called “rat anti-kinesin-2.” Secondary antibodies are raised against the constant region of the primary antibody (i.e. secondary antibodies bind to primary antibodies!) Continuing with the example above, a rat will not produce antibodies against itself, so a different host organism must be used to make antibodies against the rat constant domain. If the host organism was a chicken, this secondary antibody would be called “chicken anti-rat.” In this two-antibody approach, the reporter molecule will be attached to the secondary antibody. Direct Visualization: Alternatively, it is possible to use a primary antibody with a reporter molecule attached directly to it. In today’s procedure, a single HRP-conjugated antibody will be used to bind to our protein of interest. It is a “one and done” process! McCarville, Garant and Tatar (2023/2024) Western Blotting Overview: Recall from Lab 3 that the cell lysate proteins from the stressed and unstressed cells were separated on an acrylamide gel. When electrophoresis was complete, you transferred the proteins from the gel onto the surface of a piece of nitrocellulose membrane. During the western blot procedure today, you will use antibodies to target your protein of interest. The antibodies we are using today are conjugated to the reporting molecule horseradish peroxidase (HRP). In the presence of the chemical luminol and an oxidising reagent, this enzyme will catalyse a reaction that releases light. We cannot see the light reaction with our eyes, but we can capture the light on film, or with a camera system. Based on the intensity of the light, we gain information regarding the expression level of the protein. Based on the location of the light, we gain information regarding the molecular weight of the protein. Today you can choose either Hsp27 or p53 to be your target protein. Hsp27: This 27 kDa small heat shock protein is a molecular chaperone that protects proteins from misfolding during cell stress. It appears that Hsp27 has an important role in cell survival by regulating the activity of multiple proteins involved in apoptotic pathways. p53: This 53 kDa protein is known as the “Guardian of the Genome” because of its important role as a tumour suppressor. If a cell has experienced DNA damage, p53 will activate transcription factors that will eventually lead to cell cycle arrest, which gives the cell time to repair the damage. If the damage is irreparable, p53 will direct the cell towards an apoptotic pathway. Procedural checks: The purpose of western blotting is to determine the expression levels of your target protein. But what if the luminol/oxidizing reagent were expired? Or what if you had accidentally loaded more total protein into one lane than the other lane? Or, what if the proteins did not transfer evenly from the gel onto the nitrocellulose? To be confident that any noticeable differences in expression are valid, it is good practice to use procedural checks. Researchers use “house-keeping” proteins for procedural checks, as their expression is predictably constant. These are proteins that we would not expect to be up or down-regulated in response to our experimental conditions. Tubulin and actin are often used. We carry out a simultaneous western blot to probe for the housekeeping protein. Based on the band intensities of the house-keeping protein, we can feel confident that the protein concentrations were correct, and that there were no issues with the western blot reagents and procedure. For example, if we do not see a band for our house-keeping protein in our procedural check, that would suggest that there was something wrong with the blotting procedure. Antibodies we will use in the lab today: Mouse anti-tubulin conjugated to HRP Mouse anti-Hsp27 conjugated to HRP Target Protein Tubulin Hsp27 Rabbit anti-p53 conjugated to HRP p53 What is the purpose of doing it? This is a procedural check. Determine if the target protein is up or down-regulated in response to experimental conditions. McCarville, Garant and Tatar (2023/2024) Western Blotting Protocol: Transfer (already completed last lab!) As a reminder, last week you ran the gel, removed it from between the plastic plates, and transferred the proteins from the gel onto a piece of nitrocellulose membrane using the Turbo-Blot machine. At the end of the transfer, you retrieved the membrane (which now is coated with the proteins!) and stored it in the fridge in a plastic dish with TBS-Tween buffer. Ponceau Stain and Blocking: 1. Today we start by confirming that the cell lysate proteins successfully transferred to the nitrocellulose membrane by doing a quick stain with Ponceau Red. Wearing gloves, dispose of the buffer that is in the tray with the membrane into the blue sink at your bench, and then pour the entire vial of Ponceau Red onto the membrane. Wait ~45 seconds and pour the stain back into the vial (use the tiny funnel to avoid spillage!) 2. Use the squirt bottle on your bench to rinse the membrane several times with water. You can pour the rinses into the blue sinks on your bench. Looking at your membrane, you should see the pre-stained marker bands, as well as the cell lysate protein bands! Yay!! 3. Remember that each pair is going to probe for TWO proteins today: tubulin (the procedural check) and either Hsp27 or p53. You have already prepared for this by loading your samples in duplicate (remember, you loaded the samples in the order indicated to the right!) 4. Examine your Ponceau-stained membrane. Identify the top, where the proteins were loaded, and identify the various lanes. Use the scissors on your bench to cut the membrane into two pieces, such that each piece contains a marker, negative control and a treatment lane. One piece will be probed with the tubulin antibody and the other piece will be probed with the Hsp27 or p53 antibody. The Ponceau Red stain is temporary and will quickly wash away. Therefore, you should use a pen to label key information on the membrane. Include: • Your initials on the top left corner. • The name of the protein you are probing for (tubulin or Hsp27 or p53). • The sample that was loaded into the lane (“M” or “C” or “V” or “H2O2”). 5. Once the membranes are labeled, place them into the blue dish. Pour enough 5% milk solution (located on the side bench) so that the membranes are submerged. Set the dish on the shaker and set a timer for 25 minutes. The milk protein (called casein) will coat the nitrocellulose to reduce non-specific binding of the antibody to the membrane. 6. At the end of 25 minutes, decant the milk solution into one of the large sinks in the room (don’t use a blue sink at your desk as the milk will eventually get stinky!) 7. Pour a small bit of TBS-Tween onto your membranes. Give the dish a quick swirl and decant the TBS-Tween down the large sink. McCarville, Garant and Tatar (2023/2024) Antibody Incubation: On the side bench there are three dishes of diluted antibodies: an anti-tubulin antibody, an anti-Hsp27 antibody, and an anti-p53 antibody – all conjugated to the reporter molecule HRP. The tubulin-HRP antibody is diluted 1:10000. Both the Hsp27-HRP antibody and the p53-HRP antibody are diluted 1:5000. 1. Put each membrane into the appropriate dish and set a timer for 15 minutes. (We generally allow at least 15 minutes for this step, and sometimes, much longer! It depends on the amount of target protein present and on the antibody itself (some need more time to bind efficiently.) 2. When the antibody incubation is complete, retrieve your membranes, and put them into separate blue dishes. Pour enough TBS-Tween onto each membrane so that it is submerged. Gently agitate the dish for 2 minutes. This will wash away any unbound antibody. Pour the TBS-Tween down the blue sink. 3. Do two more 2-minute washes (for a total of 3 washes.) Finally, do a 3-minute rinse in TBS buffer (different than TBS-Tween!). We do this because the Tween detergent interferes with the chemiluminescence procedure, which is our next step! Chemiluminescence and Film Exposure: The last step is to detect the location of the antibody that is binding your protein of interest. At the side bench, pipette 200 µl of luminol and 200 µl of oxidising reagent into the same microcentrifuge tube. Make sure to change tips between reagents. With the assistance of the Instructor or TA, place your membranes side-by-side on a piece of plastic wrap and pipette the luminol/oxidizing reagent mixture onto the membrane. The HRP on the antibody will catalyze the oxidation of luminol, causing the release of light. The glow is not intense enough to be detected by the naked eye, so alternative methods must be used! There are automated systems that can read the light exposure and generate an image, but we use blue autoradiography film. In a darkroom, the light signal from HRP is captured on film – resulting in black lines on the blue film. Western Blot Analysis: Finally! We have a piece of film with bands! Any location on the film that was exposed to light will turn black. More light intensity will result in a darker and thicker band. (Note: In the western blot to the right, the varying intensities are quite obvious!) At this point we usually do three things: 1. We often compare the film to the nitrocellulose to see the location of the bands in relation to the positions of the pre-stained molecular weight markers on the nitrocellulose. i.e. are we detecting protein bands at the expected molecular weight(s) of the proteins we probed for? 2. We look at the band intensity (i.e. protein expression) of our procedural checks to determine if we performed the experiment correctly. 3. We look at the band intensity (i.e. protein expression) of our target protein in the negative control to see the “baseline” expression level. Then we look at the band intensity of the target protein in the treated sample to determine if our experimental conditions resulted in any changes in protein expression. The language that we use is “up-regulated” or “down-regulated”. In our experiment, we will compare the expression of the target protein in the stressed cells to its expression in the non-stressed cells. Before you leave the lab today: • • Clean-up your bench. Make it look the same as when you arrived! Complete and submit the In-Lab Worksheet. McCarville, Garant and Tatar (2023/2024)