Lab4-Western+Blot

Lab 4-2 Overall, SDS-PAGE followed by Western blot is a powerful tool for separating and detecting a specific protein. During electrophoresis, the SDS denatured proteins migrate through the gel towards the positive electrode at a rate that is inversely proportional to their size (MW in daltons). In other words, the smaller the protein, the faster it migrates. This is because the polyacrylamide gel serves as a maze for the proteins to travel through and smaller proteins move faster. The molecular weight of an unknown protein is obtained by the comparison of its position on the gel after electrophoresis to the positions of a standard SDS protein ladder. Figure 1 shows the process of protein denaturation and SDS gel electrophoresis. The second step is Western Blot Analysis and it involves the direct transfer of protein from a polyacrylamide gel to a charged nitrocellulose membrane. It is advantageous to transfer proteins to a membrane because: a) membranes are stronger and easier to manipulate than gels. b) proteins embedded in gels are difficult for the antibody we use later to access. By pulling the protein out of the gel and onto the membrane, the protein is more accessible to the antibody we will be using. The charged nitrocellulose membrane binds proteins with a high affinity, and proteins can migrate out of the gel and onto the sheet. After transfer, all the proteins that have been separated by size can be visualized by staining the membrane with a non-specific protein dye, such as bromophenol blue. However, a better way to detect a specific protein of interest is by using an antibody-coupled method called immunological detection . Antibodies are highly specific molecules that bind to only one type of protein. In lab four, we will attempt to detect the horseradish peroxidase we purified in lab 3. For immunological detection, the membrane is placed in blocking buffer which contains detergents and blocking proteins that bind to all unoccupied sites on the membrane. The membrane has a limited protein-binding capacity, so by saturating the sites with a non-specific protein, such as BSA, it ensures that no accidental contamination of the membrane occurs. The membrane is then incubated in a buffer that contains an antibody to the blotted target protein, HRP. The primary antibody binds specifically to HRP. Subsequent washings will remove excess, unbound antibody. A secondary antibody against the primary antibody is then applied. This secondary antibody recognizes the primary antibody and is often linked to an enzyme such as alkaline phosphatase (AP) for detection. It is critical that all non-bound secondary antibody is washed off, to be sure that the detection is specific to secondary antibody that is bound to the primary antibody. Finally, the membrane is incubated with a solution containing the substrates for AP that develops a reddish color when it interacts with the AP on the secondary antibody. A schematic diagram of a Western Blot can be seen in Figure 2.

Lab 4-3 4A. SDS-PAGE OF PROTEINS In lab 4A, we will separate proteins from a mixture of proteins by size using SDS polyacrylamide gel electrophoresis (SDS-PAGE). Our hope is that HRP will be among the proteins we separate in the gel. You will load two samples onto the gel: your CH and one fraction that shows high potential for HRP (ask your instructor which sample would be most ideal. In addition to these two samples, the instructor or a student volunteer will load two other samples onto the gel: a pre-stained protein standard containing a mixture of six different proteins of known size and a positive control sample of purified HRP that was purchased from a supply company. The figures below show the banding pattern of the protein standards and the HRP control. As you can see in Figure 3b, HRP has a size of around 44kDa.

Lab 4-5 LOADING PROTEIN SAMPLES ONTO THE GEL (10 min) 1. Using a fresh fine tip micropipette tip, MEASURE 20 µL of the first sample as indicated in Figure 5. An instructor or a student volunteer will load lanes 5 and 6. Lane 5 is the protein standard markers. This sample contains known markers with known sizes as follows in kilo Daltons (kDa): 120, 85, 50, 35, 25, and 20. Lane 6 contains a positive control of pure horseradish peroxidase. 2. PLACE the pipette tip under the buffer and directly above the sample well, resting gently against the back plate of the gel cassette. 3. Slowly DISPENSE the sample by depressing the plunger. 4. REPEAT steps 1-3 for remaining samples, changing the tip between each new sample. Be sure to change pipette tips between loading each sample! RUNNING THE GEL (90 min) 1. Once all samples have been loaded, carefully PLACE the cover onto the electrode terminals. 2. CONNECT the electrical leads to the power supply. 3. SET the voltage of the power supply to 125 volts and PERFORM electrophoresis for a minimum of 60 minutes but optimally 75 minutes (ask your instructor about the timing). Allow the proteins to separate on the gel for the recommended length of time, or until the tracking dye reaches the bottom of the gel. (Note: When the current is flowing, you should see bubbles forming on the electrodes.) 4. About halfway through the electrophoresis run, pre-wet the blotting membranes and filter papers as per the directions for electro-blotting in step 1 on p. 7. 5. After the electrophoresis is finished, TURN OFF the power supply, disconnect the leads, and carefully REMOVE the cover.

Lab 4-6 6. REMOVE the gel cassette from the electrophoresis apparatus and BLOT off excess buffer with a paper towel. 7. REMOVE the front plate in the following way: a. Lay cassette down on the table and locate the gap between the front and back plates on the corners of the cassette. b. Use a screwdriver, or a similarly thin, flat object, to torque the plates apart at the upper left corner. c. Repeat in all 4 corners of the plate and, if necessary, in the center of the left and right sides of the cassette. d. Continue to repeat b and c until the 2 plates loosen apart. e. Carefully remove the top plate. 8. PLACE the gel in transfer buffer and carefully REMOVE the gel from the back plate. SOAK for 10 minutes. The gel is now ready for the Western Blot Procedure. 4B. WESTERN BLOT PROCEDURE TRADITIONAL (NON ELECTRO-) BLOTTING 1. Pre-soak wick, blotting paper, and nitrocellulose membranes in transfer buffer for 5-10 min. When handling nitrocellulose membranes, be sure to handle them by their edges. Carefully slide the membrane out of the blue protective covers and transfer using forceps to the transfer buffer. 2. Place large gel plate on top of a container approximately 16 x 9 x 4 cm (L x W x D). Add transfer buffer to the tray and place presoaked wick onto gel plate such that ends are submerged in 2 cm of transfer buffer. 3. Place gel flat on top of wick. Smooth over top of gel to remove air bubbles. An easy way to smooth the gel over is to use a large pipette tip as a roller. 4. Place nitrocellulose membranes on top of each gel. 5. Place the two pieces of blotting paper (from step 1) on top of the membrane. Smooth over to remove all air bubbles underneath. An easy way to smooth the gel over is to use a large pipette tip as a roller. 6. Place a 6 cm stack of paper towels on top of blotting paper. Finally, place a 1 kg weight on top of stack to complete assembly, as shown in Figure 6. Now is a stopping point to let the transfer take place overnight (12-15 hours). QUESTION 1. Why are the electrophoretically fractionated proteins transferred to a membrane for immunological detection? To make proteins immobile for antibody staining and detection , the electrophoretically separated proteins are moved to a membrane . Since the proteins can still permeate through the get's pores , they are not sufficiently immobilized in the get . But after being moved to the membrane , they become fully immobile , making it possible to detect particular proteins when an antibody is added . Additionally , there is a significant improvement in the protein-antibody binding efficiency On the membrane .

Lab 4-8 QUESTION 2. Why is the membrane blocked with blocking buffer before incubation with primary antibody? 3. Obtain 10 mL of primary antibody solution which has been prepared by your instructor and INCUBATE the membrane in this solution for one hour at room temperature on a rotating or shaking platform. DISCARD the primary antibody solution. The primary antibody consists of rabbit anti-HRP antibody. It is designed to bind specifically to HRP on the membrane. 4. WASH the membrane for 5 minutes in 10 mL blocking buffer. DISCARD the blocking buffer. REPEAT this wash one more time. 5. Obtain 10 mL of secondary antibody solution which has been prepared by your instructor and INCUBATE the membrane in this solution for one hour at room temperature on a rotating or shaking platform. DISCARD the secondary antibody solution. 6. WASH the membrane for 5 minutes in 10 mL PBS buffer. DISCARD the PBS. REPEAT this wash one more time. 7. ADD 10 mL substrate solution which has been prepared by your instructor. INCUBATE for 5-10 minutes or until color development is observed. DISCARD the substrate solution. 8. WASH membrane with water and then air dry on a paper towel. Alternatively, you can blot the edge of the membrane onto a paper towel. 9. COMPARE the size of the samples containing the various concentrations relative to the protein standard markers. QUESTION 3. Why must the excess unbound secondary antibody be washed off the membrane prior to incubation with Ap substrates? The membrane is blocked with blocking buffer before incubation with primary antibody is to prevent non-specific binding of antibodies to the membrane . The excess unbound secondary antibody should be washed off the membrane prior 10 incubation with Ap substrates removes unbound or aggregated proteins present on the blots as well as unbound agents that may interfere with the detection of the target molecules .

Lab 4-9 QUESTION 4. What is the advantage of performing a Western Blot over visualizing proteins using a total protein stain? QUESTION 5. In the space below, attach or embed a picture of your gel and then describe your results. Did you get the expected results? Were there errors? Explain the banding pattern in each lane (including your two lanes, the standard lane and the positive control lane). How large was the HRP in the your lanes and the positive control lane? If they were different, explain why. The benefit of utilizing a Western Blot as opposed to a total protein stain for protein visualization is that the former enables more precise protein detection through the use of specialized antibodies directed against the target protein . It is challenging to identify the precise protein we want to visualize as total protein stain , stains every protein in a sample . Western Blot is far more sensitive technique for identifying certain proteins since it uses antibody probes . Peroxidase bands were visible in the crude homogenate samples of all the groups . Nevertheless , very few of the fractions had outcomes that were comparable to or anticipated . Since the crude homogenate is the least treated sample , even though it contains peroxidase , it does not show purity . Regarding our fraction specifically , there are not any bands that match peroxidase . This , in my opinion , is a sign of an unsuccessful experiment . It is difficult to differentiate the bands for the standard , while the positive control exhibits a single band ,