Western Blot with ECL

I. Purpose:

Western blotting is a technique used to probe for specific proteins in tissue lysates.  The proteins in the lysates are first separated by size using reducing SDS-PAGE and then transferred to nitrocellulose membrane.  The membrane is then probed with primary antibodies specific to the proteins of interest followed by enzyme conjugated secondary antibodies.  Bands are visualized by adding an enzyme substrate which forms an insoluble, colored product corresponding to the location of the protein band.  The blots can be scanned and band densities determined for semi-quantitative analysis.  

 

II. Reagents and Equipment: 

      • Molecular Weight Markers, 
      • 30% (w/v) Acrylamide
      • 1.5M Tris pH 8
      • 1M Tris pH 6.8
      • 5X Reducing Sample Buffer (0.25M DTT, 5% SDS, 0.2M Tris pH6.8, 1.5% Bromophenol Blue, 37.5% Glycerol)
      • 20% (w/v) SDS
      • TEMED (Biorad, cat #161-0801)
      • 10% (w/v) Ammonium Persulfate
      • BSA
      • Coomassie Blue Stain (0.15% Coomassie Blue, 50% Methanol, 10% Acetic Acid)
      • 5% Blotto Block Buffer (5% Skim milk powder, 0.05% Tween-20 in PBS) **No Azide for HRP conjugated secondary antibodies
      • 5% BSA antibody solution (5% BSA,0.05% Tween-20 in PBS)
      • PBS/Tween-20 Wash Buffer (PBS + 0.05% Tween-20)
      • SDS-PAGE Running Buffer (0.025M Tris, 0.192M Glycine, 0.1% SDS)
      • Transfer Buffer (0.025M Tris, 0.192M Glycine, 20% Methanol)
      • BioRad MiniGel Glass plates, electrode assemblies, blotting sandwiches, blotting sponges and electrophoresis tanks
      • PVDF membrane
      • Thin blotting paper
      • Ponceau S (Sigma, cat #P7170-1L)
      • Microcentrifuge Tubes
      • Heating block at 95°C
      • BioRad Gel Doc Imaging System
      • Primary Antibodies
      • Horse Radish Peroxidase conjugated Secondary Antibodies
      • ELC Substrate – warmed to room temperature (GE Healthcare, cat# RNP2132)
      • Kodak Film X-OMAT Blue XB (PerkinElmer  #NEF596)
      • Photo cassette BioMax

III. Assembling the Gels

      1. Once the stacking gel has polymerized, insert the electrode assemblie(s) + gel(s) into the electrophoresis tank.  If only one gel is being run, place an empty gel clamp into the other side of the electrode assembly.
      2. Fill the inner chamber with SDS-PAGE Running Buffer and ensure the chamber doesn’t leak, then fill the outer chamber with SDS-PAGE Running Buffer.
      3. Remove the comb(s)  

IV. Loading Molecular Weight Markers and Samples

      1. Using gel loading pipet tips, load the required amount of molecular weight markers( 7ul) and samples in designated wells.  Use the Western Blot Template on page 6 to keep track of sample placement and blot conditions.  
      2. If a single gel is being used for more than one antibody blot, it is convenient to leave a blank well between sets so there is plenty of room to cut the blot later without cutting through samples.
      3. Ensure that enough Molecular Weight Marker is loaded (protein-wise) so that all bands can be visualized with Ponceau S after transfer.
      4. Avoid pipeting bubbles while adding samples to wells as this may cause samples to bubble out of the wells.

V. Running the Gel

      1. Once all samples and molecular weight markers are loaded, place the electrophoresis chamber lid on and connect to a power supply.
      2. A guideline is to run the gel at 90V for 10 minutes through the stacking gel and then at 120V through the separating gel.  Stop applying voltage once the dye front is near the bottom of the gel.

VI. Transferring Proteins from the Gel to the Nitrocellulose Membrane

      1. Activate the PVDF membrane by immerse it for 10sec into methanol and afterwards washing the membrane for 1min in water before you soak it for 15min in transfer buffer   
      2. Pre-wet the blot sponges and filter paper in 4°C Transfer Buffer.  **Never handle PVDF membrane with bare hands or even gloves.  The preferred method is to use tweezers.
      3. Remove the gels from the electrode assemblies one at a time being careful not to tear them.
      4. Assemble the blot sandwiches one at a time as shown in Figure 1.
      5.  Insert the blot sandwiches into the blot electrode assembly as well as blue ice pack which is kept in the freezer.
      6. Fill the electrophoresis tank with cold Transfer Buffer and add a stir bar.
      7. Perform the transfer on a stir plate at 4C.  Run at 40v over night.
      8. After transfer is completed, remove the blot sandwiches and take them apart.  
      9. If desired, stain the nitrocellulose membrane with Ponceau S for a few minutes.  
      10. Remove the Ponceau S stain by washing the blot a few times in PBS/Tween-20 wash buffer.
      11. After washing, incubate the blots in 5% Blotto Blocking Buffer for 1h.
      12. Wash blots in PBS/Tween-20 Wash Buffer 3 x 5 min on the Belly Dancer 
      13. If desired, the SDS-PAGE gel can be stained with coomassie blue to determine the success of the transfer.  You will expect to see some protein remaining in the gel but it should be quite faint. Therefore incubate gel with Coomassie Blue in a staining tray for 15 min then pour off the stain back into the stock bottle (it can be re-used). Destain the gel by filling the staining tray with water and a rolled up large kimwipe.  Microwave for approximately 1-2 minutes…it should get very hot…close to boiling.  Remove from the microwave and put on the Belly Dancer for 5-10 minutes.  Pour off the old water and add fresh.  Repeat steps 13 to 15 a few times until it has been destained sufficiently.  

 

VII. Probing the Blot with Antibodies

      1. Prepare the primary antibody at dilutions recommended by the vendor at dilutions tested in-house and found to be successful.  Dilutions are made in 5% BSA in PBST.  In general, the volume required for an entire 7.5 x 10cm piece of nitrocellulose is approximately 8-12mL.
      2. If necessary, the PVDF can be cut into sections for incubation with different antibodies.  A razor blade on a hard surface works well for this.  Try and be quick so that the blot doesn’t dry out.  It is important to mark each section with ballpoint pen to keep track of what antibody is used on what section.  Put the blot back into Wash Buffer after cutting so it doesn’t dry out.
      3. Prepare the antibody incubation by fill the antibody solution into a 15ml or box tube ( Don’t forget to label tube, antibody can be used multiple times therefore mark down how often it has been used)
      4. Insert membrane with tweezers into the tube or box (Hint: Bend the sides a bit) and incubate on the Belly dancer for 1-3 hours at room temperature, or overnight at 4C.
      5. Wash blots in PBS/Tween-20 Wash Buffer 3 x 5 min on the Belly Dancer.
      6. Prepare the secondary antibody dilutions in 5% Blotto Blocking Buffer minimum of 8-12ml per membrane is required.
      7. When the blots are washed, insert them into 15 ml tube or box filled with 8-12ml of antibody solution and incubate for 1h at room temperature on the Belly dancer. 
      8. Wash again as in step 5.
      9. Detect the signal with ECL substrate in the dark room in the petch building on the top floor
      10. Start the photo developer by opening the water valve, switching the machine on and closing the lid of the machine which is just laid a little bit of to the side. The developer needs to heat up for at least 5min before you can start to develop.
      11. Mix 2ml of solution A with 50ul of solution B 
      12. Lay membrane into a plastic wrap and incubate with 1ml ECL substrate per membrane for 5min (either in the dark or light is fine). 
      13. Place membrane into the photo cassette between the plastic films (can be performed in the light)
      14. Switch of the light and take one photo film, snip of the right corner to remember the orientation and lay into the cassette, close the cassette for 10, 30 sec or 1 to 3 or even 10 min if the signal is really weak.
      15. Remove the membrane immediately and insert it into the developer (if it does not feed in by itself push button in the front). 
      16. Clean the developer by washing the developer and fixer rollers and turning of the water valve as well as leaving the lid partially open. 

VIII. Scanning the Blot

      1. Turn on the BioRad Gel Doc Imaging System and open up the Quantity One Software.
      2. Insert Kodak film onto with white tray.
      3. For imaging blots, use the UV to White light converter screen and the epi-white light source.
      4. Acquire image and annotate to mark the molecular weight markers and lanes if desired.
      5. Save image and print a copy for your notebook.