MCB 253 - Western Blot

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University of Illinois, Urbana Champaign *

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253

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Biology

Date

Apr 3, 2024

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pdf

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Uploaded by DeanLemur3560

Kaela Maghinang & Anushka Singh MCB 253 March 7, 2024 WESTERN BLOTTING PROTOCOL Background: Purpose : As said by a publication by Rasheed Sule, Gabriela Rivera and Aldrin V Gomes, “Western blotting, also known as immunoblotting, is one of the most commonly used techniques in molecular biology and proteomics in scientific laboratories around the world today” (Sule, et al 2023). By utilizing this lab technique, we are able to identify our unknown protein from a composite blend of other proteins as well as that unknown protein’s molecular weight. First, we must prepare our SDS-PAGE gel using the procedure from last week’s lab. Then, we can transfer our proteins run on the gel onto a nitrocellulose membrane by using an electrical current and filling the chamber with a blotting buffer. Afterwards, primary and secondary antibodies are used to stain the nitrocellulose membrane and thus, visualize which antibody was bound to our unknown protein. This gives us additional information as to which of the given proteins match our unknown protein. By employing the aforementioned techniques (Western blotting, SDS-PAGE Gel Electrophoresis, antibody staining), the hypothesis of this experiment is that whichever nitrocellulose membrane contains a dark band, the antibody used to stain that nitrocellulose membrane tellus us the identity of our unknown protein. Protocol: SDS-Page Protocol: 1. Perform SDS-Page protocol and fill the gel according to the following table: Lane 1 2 3 4 5 6 7 8 9 10 Volume 5 μL of MW standard 6 μL PBS 6 μL SB 5 μL unknown protein 1 μL PBS 6 μL SB 5 μL of MW standard 6 μL PBS 6 μL SB 5 μL unknown protein 1 μL PBS 6 μL SB 5 μL of MW standard 6 μL PBS 6 μL SB 5 μL unknown protein 1 μL PBS 6 μL SB 5 μL of MW standard 2. Run the gel at 200V for around ~30-45 minutes, remove using opening tool and place onto a weigh boat Western Blot Protocol - Transfer 1. Obtain your SDS-Page stained gel in weigh boat. 2. Fill the weigh boat with enough blotting buffer to cover the gel. 3. Trim the gel between each MW standard lane and soak it in the blotting buffer for 15 minutes. 4. Soak the sponges and filter sheets in the blotting buffer. 5. Take the tray, making sure that the black side is facing downwards in the buffer.

6. First, place the sponge on the tray. Then, place the white filter paper on top, making sure there are no air bubbles between the sponge and the filter paper. 7. Then, place your SDS-Page gel on top, again, making sure there are no air bubbles between the gel and the filter paper. 8. Take off the protective sheet on top of the nitrocellulose membrane and soak it in the blotting buffer. 9. Place the nitrocellulose membrane on top of the gel. This should not be moved after as proteins will begin to blot immediately. 10. To remove any air bubbles between the membrane and the gel, and take a 1000μL pipette tip and roll on top of the membrane. 11. Place another filter paper on top of the membrane, followed by another sponge (note that these should be soaked in blotting buffer) 12. Close the cassette tray by folding the clear plastic side to the black, making sure that the layers are pressed together for transfer to be successful. Lock the cassette tray in place. 13. Place cassette tray into the chamber with an ice pack and add blotting buffer until the black line labeled “4 gel line” or “blotting line” 14. Run the chamber at 100V for 45 minutes 15. Once done, open the chamber and take out the cassette. Open the cassette and remove each layer until you get to the nitrocellulose membrane. Remove the membrane and leave it to dry. Western Blot Protocol - Detection/Staining 1. Obtain the dried nitrocellulose membrane and trim between lanes 3 and 4 as well as lanes 6 and 7. 2. Place each part of the membrane into separate weigh boats and pour 25 mL of blocking buffer into each weigh boat. Place these onto a rocker for 15 mins. 3. Discard the blocking buffer and place 10 ml of working buffer and 10μL of your primary antibody into each weigh boat. Place onto rocking tray for 30 mins a. We chose to use three separate primary antibodies to identify our unknown protein. In particular, we will use anti β-galactosidase antibody produced in mouse, anti-myosin light chain antibody produced in mouse and anti-Bovine Serum Albumin (BSA) antibody produced in mouse. 4. Discard the solution in each weigh boat and wash excess primary antibodies with working buffer. Wash these 3 times for 5 minutes each onto the rocker. 5. Discard the working buffer in each weigh boat. Add 10 mL of working buffer and 10μL of secondary antibody into each weigh boat. Place onto the rocker for 25 minutes a. We chose to use only one secondary antibody as all of our primary antibodies were produced in mice, therefore, our secondary antibody is Goat anti-mouse conjugated HRP. 6. Pour off the secondary antibody solution and wash each weigh boat with working buffer 3 times for 5 minutes each onto the rocker. 7. Add 10 ml of HRP color development solution to each weigh boat and cover with foil to allow bands to develop. 8. Once bands appear, rinse with 10 mL of distilled water in each weigh boat 3 times for 5 minutes each onto the rocker. 9. Discard the distilled water and let each membrane air dry. Take photos of each membrane and collect data.

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