Western blot protein detection using chemiluminescence

Last updated: 1/12/12 By: Tim Starr

Overview

Follow this protocol to detect proteins on a PVDF or nitrocellulose filter. The first step is to "block" the membrane, which means to soak the membrane in a buffer containing a lot of miscellaneous protein (like albumin in milk or Bovine serum albumin). The purpose is to cover any part of the membrane that will bind non-specifically to proteins, that way when you add the antibody specific to your protein it will only bind to your protein and not to other parts of the membrane by accident. The second step is to soak the membrane in an antibody that is specific for the protein you want to detect. The third step is to soak the antibody in a secondary antibody that binds to the primary antibody. This secondary antibody will be attached to an enzyme such as HRP or a fluorescent molecule, which can be used for detection. The last step is to soak the membrane in the substrate solution and then image the membrane

Protocol

• Wash the membrane. Place membrane in container (plastic weigh boat, pipette tip box, seal-a-meal bag, band-aid box, etc.) and rinse 4-6 times with wash buffer (TBS-T or TBS), then add enough wash buffer to cover. Place container on a rocker for 10 minutes. Note: Can leave membrane in wash buffer indefinitely.

Wash Buffer: Tris-Buffered Saline with Tween20 (TBS-T)

• Variations to the above buffer include: Tris at 50 mM; phenol red at 40 mM (0.015 g/L); Tween20 at 1.0%
• Block the membrane. Replace the wash buffer with blocking buffer. Blocking buffer consists of the Wash buffer above plus some random protein. Commonly use 2 to 5% milk powder or bovine serum albumin powder. Can also use a solution of IgG from the same species that was used to make the antibodies you will use. Place on rocker for at least 1 hour, but can block for as long you want. If going overnight, place in cold room.
• Primary antibody staining. Pour off the blocking buffer and replace with blocking buffer plus the primary antibody. Concentrations of the primary antibody range from 1:100 to 1:10,000. Use trial and error to determine appropriate concentration. Place on rocker and incubate a minimum of 1 hr. Can incubate up to 24 hours. If incubating for a long time, generally perform the incubation in the walk-in cooler.
• Note: Can save the primary antibody buffer mixture and reuse multiple times.
• Wash the membrane with TBS-T. Rinse 4-6 times, then place on rocker for at least one hour, changing wash buffer two to three times. This can be done for a longer period of time.
• Secondary antibody staining. Replace wash buffer with 2º antibody in blocking buffer at appropriate concentration (generally from 1:1000 to 1:10,000 or 1:100,000 for monoclonal 2º’s). 2º antibody is normally conjugated either to Horseradish Perioxidase or to Alkaline Phosphatase. These enzymes will then cleave a substrate that can be detected either on film or using a scanner.
• Note: Can save the secondary antibody buffer mixture and resuse multiple times
• Wash the membrane with TBS-T. Rinse 4-6 times, then place on rocker for at least one hour, changing wash buffer two to three times. This can be done for a longer period of time.
• The membrane is now ready for chemiluminescent or chemifluorescent detection of the secondary antibody.
• Chemiluminescent detection. Different types of chemiluminescent substrates exist. Currently using SuperSignal Femto chemiluminescent substrate. Prepare chemiluminscent substrate by mixing equal parts of bottle A and bottle B. Need about 2 ml for one 3" x 3.5" gel. Incubate membrane in 2 ml of the chemiluminscent substrate for 5 to 10 min. on rocker. Note: some people just put the substrate on the membrane and let it sit on the bench.

• X-ray film: If you will use X-ray film to image the membrane, prepare film apparatus. Put saran-wrap covered whatman paper on bottom of apparatus. Place the glowing stickers on the saran wrap on the top and side of where the membrane will be placed. Place the membrane with the side that was facing the gel facing up. Place another piece of saran wrap on top of the membrane. Take the film apparatus to dark room. In the dark room, turn off lights, place a sheet of film over the membrane (try to do this smoothly, without moving the film or membrane) and cover for appropriate length of time (1 second to 1 minute to longer). Put film in the developing machine. Note: The chemiluminescent stuff will gradually get brighter for about a half hour and then gradually get darker, so the developing time is really an artistic guess. Wait to see if picture develops. Take another if it didn’t come out well.

• ProteinSimple digital imager: If you will use the ProteinSimple digital imaging machine to image the membrane. Place black screen on imaging box. Place membrane inside the square on the black screen. Follow ProteinSimple imaging instructions for taking the digital image.