Western Blot Protocol

Western blotting provides a semi-quantitative method to measure protein levels. This immunoblotting method detects the presence and relative quantity of proteins, their molecular weight, and the efficiency of protein extraction. The Western Blot technique is composed of 4 steps: 1) separation of proteins by gel electrophoresis, 2) transfer of proteins to a solid support, 3) detection of target protein, and 4) visualization of target protein. After the transfer step, the membrane undergoes a blocking step which covers the entire surface of the membrane with detergent solution to prevent non-specific binding of antibody to the membrane. Next, the membrane is incubated with a primary antibody which binds to the target protein on the blot, followed by a conjugated secondary antibody which binds to the primary antibody. Finally, the protein is visualized by the addition of a reagent which reacts in the presence of the label on the secondary antibody.

Materials:

• SDS-PAGE materials
• Transfer buffer: 0.04% SDS, 20% Methanol, 48 mM Tris, 39 M glycine
• Nitrocellulose membrane
• 6 sheets of absorbent filter paper
• Transfer apparatus
• Washing buffer: PBS with 0.1% Tween ®20
• Blocking solution: 3-5% BSA in PBS with a small amount of Tween®20
• Orbital shaker platform
• Appropriate primary and conjugated secondary antibodies
• Reagent for visualization of conjugate

  I. Gel Electrophoresis:

1. Run an SDS-PAGE to separate the proteins on a gel.

 II. Protein Transfer:

1. Cut a sheet of nitrocellulose paper and 6 sheets of absorbent filter paper to the exact size of the SDS-PAGE gel.
2. Pre-wet the nitrocellulose membrane in deionized water. Wear gloves when handling the membrane.
3. Soak the gel, nitrocellulose membrane, and absorbent papers in transfer buffer for 5 minutes.
4. Apply the membrane to the surface of the gel. Remove any air bubbles by rolling a pipette across the membrane.
5. Sandwich the gel, blotting membrane and absorbent papers as follows: bottom plate (cathode) - 3 sheets of absorbent paper – gel – membrane  – 3 sheets of absorbent paper – top plate (anode). Verify the orientation of the gel and membrane to ensure that the proteins migrate in the correct direction; the membrane should be above the gel, facing the positive electrode.
6.  Connect the electrodes. Run the transfer for 45 minutes to 1.5 hours with a current of 0.8 mA/cm².
7. Disconnect the power supply. Remove and disassemble the sandwich, removing the nitrocellulose membrane. A corner may be snipped off in order to retain the orientation of the membrane.

III. Detection of Target Protein

1. Wash the membrane twice for 5 minutes with deionized water to remove gel and transfer buffer components and weakly bound proteins.
2. Incubate the membrane in blocking solution for 30 minutes to 2 hours at room temperature with gentle agitation.
3. Rinse the membrane with PBS twice for 5 minutes. 
4. Dilute the primary antibody in blocking solution to a concentration between 1 µg/ml and 50 µg/ml. (Eg.  add 10 µl of a 1 mg/ml antibody to 10 ml of blocking solution for a 1 µg/ml dilution.) Incubate for a minimum of 1 hour at room temperature or overnight at 4°C with gentle agitation.
5. Wash the membrane for 5 minutes with PBS. Repeat wash 3 times.
6. Prepare a dilution of the secondary antibody in blocking solution. The concentration should be between 0.5 to 5 µg/ml, or a dilution of 1/200 to 1/2000 of a 1 mg/ml stock. Incubate the membrane in the secondary antibody solution for 1 hour at room temperature.
7. Wash the membrane for 5 minutes with PBS. Repeat 3 times.
8. Drain the reagent and blot the membrane on a clean piece of paper towel.

 IV. Visualization of Target Protein

1. Immediately prior to use, prepare fresh reagent for visualization and develop the blot. For example, with an alkaline phosphatase labeled secondary antibody, add 66 µl of NBT stock to 10 ml of alkaline phosphatase buffer with mixing, then add 33 µl of BCIP stock. Add 10 ml of solution per 15X15 cm² membrane. Develop the blot at room temperature until the bands have darkened (approx. 30 minutes). Stop the reaction with PBS containing 20 mM EDTA.

References:

Using Antibodies: A Laboratory Manual. Harlow & Lane. Cold Spring Harbor Laboratory Press. 1999.

Introduction to Antibodies, 2^nd Edition. Chemicon International.

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