Western Blot Instructions

Western blot analysis is a well-known technique for identifying specific proteins isolated from tissue. The different proteins are first separated by charge and weight on SDS-Polyacrylamide gel and transferred to a nitrocellulose membrane. After transfer, labeled antibodies bind specifically with the desired protein and are photographed using X-ray film or visualized by protein-specific stain. The final result shows dark bands indicating the presence of the identified protein. This article focuses on the basic methodology for performing Western blot analysis in a laboratory qualified for complex protein research.

Things You'll Need

• SDS-polyacrylamide gel (SDS-PAGE) with separated proteins
• Nitrocellulose membrane
• Distilled water
• Transfer buffer (30 g Tris, 144 g Glycine and 200 ml methanol in 1 l water)
• Filter paper, Whatman 3 mm
• Laboratory sponge
• Western blot transfer chamber
• Laboratory power supply
• Laboratory refrigerator
• Laboratory agitator
• Tris buffered saline solution (TBS) (24.2 g Tris Base, 80 g NaCl in 1 l water)
• Blocking solution (5 percent nonfat powdered milk in TBS)
• Primary antibody solution
• Labeled antibody conjugate solution
• Alkaline phosphatase substrate solution with nitro blue tetrazolium stain
• Phosphate buffered saline (PBS)
• Gel-imager box

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Instructions

Transfer Proteins to Nitrocellulose Membrane

• 1

  Prepare the nitrocellulose membrane. The membrane should be soaked in distilled water for two minutes, then placed into the transfer buffer and allowed to soak for five minutes.

• 2

  Assemble a transfer sandwich consisting of sponge/filter paper/SDS PAGE/nitrocellulose membrane/filter paper/sponge. The sandwich is aligned so the membrane lies closer to the anode and the gel closer to the cathode inside the transfer chamber.

• 3

  Add transfer buffer in the chamber so the sandwich is submerged. Place the chamber inside the laboratory refrigerator. Transfer of protein from the gel to the membrane should be performed at 4 degrees Celsius.

• 4

  Set up the power supply to transfer proteins from the gel onto the membrane. The direction of transfer is toward the anode indicated by the red lead. Larger proteins move more quickly in response to an electrical current and will move first. The power supply controls the rate of transfer. Set the power to 30 V and 40 mA for an overnight transfer.

• 5

  Disassemble the power supply after transfer is complete. Do not make contact with the transfer buffer or remove the sandwich while the power supply is attached.

• 6

  Remove the membrane from the sandwich and incubate for two hours in blocking solution. Proteins in the blocking solution will absorb into the membrane where no proteins are present. It will prevent any antibody from binding to the membrane where the desired protein is not present.

Binding the Antibody

• 7

  Incubate the nitrocellulose membrane with primary antibody solution using an agitator. The membrane must be completely submerged in the solution for at least one hour. It is acceptable to incubate overnight.

• 8

  Wash the membrane thoroughly to remove unbound antibody. Four separate 10-minute washes with TBS are usually sufficient to remove excess antibody.

• 9

  Incubate the membrane in labeled antibody conjugate solution using the agitator. The membrane needs to be incubated for a least one hour. The labeled antibody conjugate is bound to alkaline phosphatase. It is the enzyme-antibody mix that will recognize and bind to the first antibody.

Detection of the Protein

• 10

  Rinse the membrane for 10 minutes in PBS four times. This will remove the remainder of the labeled antibody conjugate.

• 11

  Incubate the membrane in alkaline phosphatase substrate solution until band patterns appear. The dye in the substrate solution will bind to the antibody-antigen-protein in the membrane and form a dark-colored band. The process may be immediate or require up to 30 minutes before the bands are observed.

• 12

  Wrap the membrane completely in cellophane and place it directly on the gel-imager to visualize the results. The gel-imager is capable of photographing the results displayed on the Western blot membrane.