Western Blot

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1. Prepare the following solutions: SDS-sample buffer (0.05 M Tris-HCl pH 6.8, 10 % glycerol, 1 % SDS, 0.05 M DTT, 0.01% bromophenol blue); RAPID reducing buffer (20x solution: 62.8 g MOPS (free acid), 72.6 g Tris (base), 10 g SDS, 6.5 g Sodium Bisulfite, water to 500 ml) and cold blotting buffer (for 1 L: 3g Tris base, 14.4 g glycine, 200 ml methanol, 10 ml 10 % SDS, water to 1L, pH should be ~8.3)
   
   
2. After growing liquid cultures, spin down 4-5ml aliquots at 2000 rpm for 10 minutes. Remove the supernatant and resuspend the pellet in 100 uL of the SDS-sample buffer into an Eppendorf tube. Keep in the -20°C freezer for at least 30 minutes.
   
   
3. Boil the samples at 100°C for 10 minutes and spin for 1 minute at 13300 rpm. Store the samples at -20°C before use.
   
   
4. Run samples on Expedeon pre-cast gels. The 4-20 % gels are a good choice when blotting proteins of different sizes. The maximum sample volume for the 12 well gels is 35 μl and for the 17 well gels 20 μl. Load 10 μl of prestained kaleidoscope protein ladder in the first lane so that the orientation of the gel can be determined and the efficiency of blotting checked.
   
   
5. Run using 1 x RAPID reducing buffer. Gels should be run at 90 mA/gel with a maxium of 180 V; run time is 30 – 60 minutes.
   
   
6. Now, blot onto PVDF membrane using the BIORAD kit: Cut two sets each of three pieces of Whatman paper to the same size as the gel (stacking gel will be removed before blotting. Cut a piece of PVDF membrane to the size of the gel and immerse in methanol (use tweezers when handling the membrane when possible) for 5 minutes. Transfer the PVDF membrane to cold blotting buffer. Place a blotting cassette black side down in a large box of cold blotting buffer. Immerse a blotting pad and one of the sets of Whatman paper in the buffer. Place the blotting pad and then the Whatman paper onto the cassette smoothing out any bubbles with a spreader.
   
   
7. Remove the gel from the plastic casing, place in blotting buffer and then onto the cassette. Add the PVDF membrane to the cassette making sure there are no bubbles. Immerse a blotting pad and the second set of Whatman paper in the buffer. Place the Whatman paper and then the blotting pad onto the cassette smoothing out any bubbles with a spreader. Close the blotting module and place in the running tank. If only one gel is to be blotted then fill the other space with a second cassette containing two blotting pads. Insert a cooling block containing frozen Milli-Q. Fill tank with blotting buffer, place tank in an ice bucket and run at 0.5 A for 1 hour. Disassemble blotting apparatus and place blot in 5% milk powder in PBS with shaking overnight.
   
   
8. The next day, tinse the blot in PBS and then wash in fresh PBS with shaking whilst preparing primary antibodies.
   
   
9. Dilute primary antibody in 1% milk powder in PBS according to the table at the end of the protocol (serial dilutions are usually necessary in order to get a sensible dilution factor). 10 ml is required per blot. Incubate at room temperature with shaking in a clean square petri dish for at least one hour – seal the petri dish with parafilm to ensure that the blot does not dry out.
   
   
10. Rinse the blot with PBS and then wash (timings are minimum, not exact, use tweezers when moving blot): 10 mins PBS; 10 mins PBS with 0.2 % Tween 20; 10 mins PBS with 0.2 % Tween; 10 mins PBS
    
    
11. Dilute the secondary antibody in 1 % Marvel in PBS (1 in 20000 is a good starting point, if the background is too high then increase to 1 in 40000). 10 ml is required per blot. Incubate at room temperature with shaking for at least 1 hour.
    
    
12. Rinse the blot again with PBS and then wash (timings are minimum, not exact): 10 mins PBS; 10 mins PBS with 0.2 % Tween 20; 10 mins PBS with 0.2 % Tween; 10 mins PBS
    
    
13. In order to detect the proteins by chemiluminsecence, prepare the HRP (horseradish-peroxidase) substrate. For a 7 x 8.5 cm gel use 3 ml luminal reagent + 3 ml peroxide solution. For a 10 x 10 cm gel use 5 ml luminal reagent + 5 ml peroxide solution. Allow the substrate to reach room temperature. Protection from light is not required
    
    
14. Using tweezers, lift the blot from the petri dish and then touch the edge of the blot on some blue roll to remove excess liquid. Place on a piece of OHP film. Pour the HRP substrate onto the blot and incubate at room temperature for 5 minutes.
    
    
15. Lift the blot from the substrate using tweezers and touch the edge of the blot on some blue roll to remove excess liquid. Cover the blot with OHP film and remove any air bubbles.
    
    
16. Place the blot on the gel doc and focus using white light.
    
    
17. Now, make sure that the doc is set to no light and no filter. Set up multiple exposure runs each of 30s for at least 30 minutes (i.e. 60 exposures). Make sure the software is set to sum the images (you will get 60 images, each with effectively a longer exposure time). Save the best image for analysis.