Western Blotting

Western blots allow one to determine the molecular weight of a protein and to measure relative amounts of the protein present in different samples. First, the protein samples are seperated by gel electrophoresis, usually SdsPage. The proteins are then transfered to a special type of blotting paper or membrane while retaining the same pattern of seperation they once had on the gel. The blot is incubated with a generic protein (such as a milk solution) to bind to non-specific places on the blotting paper or membrane. An antibody (which usually has an enzyme or dye attached to it) is then added to the solution which is able to bind to a specific protein. Lastly, a colorless substrate is incubated with the antibody to reveal the location of the antibody which converts the enzyme (bound now to the antibody) to a colored product that can be viewed and photographed.

A one hour, high electric field strength tank-blotting transfer is very effective.

Reagents

Transfer Buffer (1L)
3.03g Tris-base
14.4g Glycine
200mL Methanol

• use good quality methanol so that metallic impurities in the methanol won't electroplate the electrodes in the transfer apparatus
  

Make up to 1 Liter volume with nanopure water and store at room temperature.

Tris Buffered Saline-with Tween-20 (1 L) (TBST)
9.9mL 1M Tris pH 7.5
1mL Tween-20
19.8mL 5M NaCl (or 5.79g)
Make up to 1 Liter volume with nanopure water and store at room temperature.

5% Milk Blocking Buffer Solution (1 L) (TBST-milk)
50g powdered non-fat dried milk
Make up to 1 Liter volume with TBST and store in the 4°C refrigerator for up to 3 days.

• note: do NOT use milk blocking solution with streptavidin detection methods. biotin in the milk will give unacceptably high backgrounds. instead use 1% BSA (bovine serum albumin - fraction V) in PBS (phosphate buffered saline). there is no biotin in BSA, but BSA is vastly more expensive than non-fat dried milk
  

Chemiluminescent Kit
ECL kit for use with secondary antibodies conjugated to either horseradish peroxidase, or alkaline phosphatase

• be sure you use the right kit for your secondary antibody!
  

Transfer of Gel to Membrane

The Mini Trans-Blot Electroforetic Transfer Cell consists of the electric module, the Bio-Ice cooling unit, gel holder cassettes containing fiber pads, buffer tank and lid. Please refer to the Mini Trans-Blot Electrophoretic Transfer Cell Instruction Manual for for pictured diagrams.

Preparation for Blotting

Fill the Bio-Ice cooling unit with water and store it in the freezer at -20'C until ready to use. After use, return the cooling unit to the freezer for storage.

1. Prepare the transfer buffer as listed above in the Reagents section.
   
2. Cut the membrane and the filter paper to the dimensions of the gel. Always wear gloves when handling membranes to prevent contamination
   
3. Equilibrate the gel and membrane separately in transfer buffer with gentle agitation for 15 minutes
   
   • this removes the salts present in the SdsPage running buffer from the gel
     
4. Prepare the gel sandwich.
   
   • Place the cassette, with the dark side down, on a clean surface.
     
   • Place one pre-wetted fiber pad on the dark side of the cassette.
     
   • Place a sheet of pre-wetted filter paper on the fiber pad.
     
   • Place the equilibrated gel on the filter paper.
     
     • make sure there are no bubbles between the gel and the filter paper
       
   • Place the pre-wetted membrane on the gel
     
     • Removing any air bubbles which may have formed is very important for good transfer. Use a glass tube to gently roll air bubbles out.
       
   • Complete the sandwich by placing a piece of pre-wetted filter paper on the membrane, again removing any bubbles.
     
   • Add the last fiber pad.
     
5. Close the cassette firmly, being careful not to move the gel and filter paper sandwich. Lock the cassette closed with the white latch.
   
6. Place the cassette in the module. Repeat the other cassette, if necessary.
   
7. Add the frozen Bio-Ice cooling unit. Place in tank and completely fill the tank with transfer buffer.
   
8. Add a standard stir bar to help maintain even buffer temperature and ion distribution in the tank. Set the speed as fast as possible to keep ion distribution even.
   
9. Put on the lid, plug the cables into the power supply, and run the blot.
   
   • for running the blot overnight, set the power supply at 30V with 90mA
     
   • for running 1 hour at high intensity field, set on 100V and 350mA
     
     • be sure to set both voltage and current limits
       
10. Upon completion of the run, disassemble the blotting sandwich and remove the membrane for development. Place the the membrane(s) in a dish containing the 5% milk blocking solution. Store @ 4°C overnight or at room temperature on the lab rotator for 30 min.
    
11. Clean off the cell, fiber pads, and cassettes and rinse well with water
    
12. Rinse off and put fresh water in the Bio-Ice cooling unit and put in the freezer so it will be ready for next time
    

Non-specific blocking

It is important to block all of the non-specific protein binding sites on the membrane, otherwise the primary antibody (a protein) will bind directly to the membrane instead of to the blotted proteins on the membrane, giving high background signal.

• Block the membrane in TBST-milk solution with gentle agitation at room temperature for one hour
  

OR

• block overnight at 4°C
  
  • the membrane will be stable in TBST-milk solution at 4°C for several days if necessary
    

Primary Hybridization and Incubation of Membrane

After the non-specific blocking step has been performed, primary hybridization will take place.

1. Pour off the used blocking milk solution and replace it with enough fresh TBST-milk solution to cover the membrane(s)
   
   • use the minimum amount of TBST-milk that will give good coverage. this will allow less antibody to be used
     
2. Add the primary antibody directly to fresh milk and membrane(s) at the appropriate concentration based upon the amount of milk in the dish
   
3. Incubate the membrane(s) at room temperature on the lab rotator for 45 minutes.
   

Secondary Hybridization and Incubation of Membrane

After incubation, pour off milk solution and briefly rinse the membrane with TBST (no milk). Add fresh TBST-milk to the dish. Add the secondary antibody directly to the fresh milk and membrane(s) at the appropriate concentration based upon the amount of milk in the dish. Incubate the membrane(s) once again at room temperature on the lab rotator but this time for 1 hour.
Most secondary antibodies are conjugated with either horseradish peroxidase (HRP) or alkaline phosphatase (AP). Make sure to remember which secondary antibody is used for there might be different chemiluminescent kits designed for either one, not both.

Development and Detection

Once the secondary incubation is completed, pour off the milk solution and rinse the membrane(s) with TBS-Tween 20 on the lab rotator to clean the blots for development and detection. Three five minute washes with TBS-Tween 20 will be sufficient. Leave membrane(s) in final wash solution until ready to develop. If not immediately ready to develop, store membrane(s) @ 4°C.

With the use of the Chemiluminescent kit, begin to develop the membrane(s).

1. put the membrane, protein side up, on an acetate sheet (blank overhead transparency)
   
2. mix 1 ml of luminol solution and 1 ml of the hydrogen peroxide (or alkaline phosphatase if that's the kind of secondary antibody conjugate you used) reagent in a 15 ml tube
   
3. butt a paper towel up against the side of the membrane and tilt the acetate sheet to drain any remaining solution from the surface of the membrane
   
   • do not let the membrane actually dry out
     
4. lay the acetate sheet with the drained membrane on it down flat and carefully layer on 1 ml of the mixed chemiluminescent solutions from above
   
   • due to surface tension, the solution will be able to sit on and cover the membrane without simply running off. you can get good exposure to the chemiluminescent reagents this way while using a lot less of these (expensive) solutions than if you agitated the membrane in a dish as in the blocking and hybridization steps
     
5. after one minute butt a fresh paper towel up against the membrane and tilt again to drain off the chemiluminescent reagents (but don't completely dry out the membrane). layer a second clean acetate sheet on top of the membrane to make an acetate sheet sandwich with the membrane in the middle. wipe a paper towel thoroughly across the top acetate of the sandwich to press out any remaining liquid chemiluminescent solution
   
6. cut down the transparency sandwich to fit onto x-ray cassettes for the developer making sure that the membrane(s) is facing up. Take the cassettes over to the dark room.
   
7. once in the dark room, make sure that the lights are turned out. Place a piece of x-ray film on top of the membrane(s) and close the cassette. Wait anywhere from 15 sec to 1 min to expose the film. Open the cassette, place the x-ray film length-wise onto the developer and it will feed through. Once a beep has been heard, other film may be fed through the developer. Try different times on film to get a good exposure.
   
   • take great care that you DO NOT FEED THE ACETATE SANDWICH THROUGH THE DEVELOPER. the acetate can adhere to the film from static electricity so you need to watch out for this
     
   • do not turn on the light at any time when in the dark room when film pack is open. The light will destroy and over expose the film!
     
   • do not get the film wet with the chemiluminescent kit reagents. These can destroy the chemicals in the developer if they are carried in on the film.
     
8. after the development of the membrane(s), the membranes may be tossed away or may be rinsed in TBS-Tween20 and then stored at 4°C if further analysis is needed.