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Western blotting uses specific antibodies to identify proteins that have been separated based on size by gel electrophoresis. The immunoassay uses a membrane made of nitrocellulose or PVDF (polyvinylidene fluoride). The gel is placed next to the membrane and application of an electrical current induces the proteins to migrate from the gel to the membrane. The membrane can then be further processed with antibodies specific for the target of interest, and visualized using secondary antibodies and detection reagents.
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These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.
Check the pH and adjust to 6.8
Check the pH and adjust to 8.3
For proteins larger than 80 kDa, we recommend that SDS is included at a final concentration of 0.1%.
3–5% milk or BSA (bovine serum albumin)
Add to TBST buffer. Mix well and filter. Failure to filter can lead to spotting, where tiny dark grains will contaminate the blot during color development.
The time and voltage may require optimization. We recommend following the manufacturer’s instructions. A reducing gel should be used unless non-reducing conditions are recommended on the antibody datasheet.
The gel percentage required is dependent on the size of your protein of interest:
Gradient gels can also be used.
The membrane can be either nitrocellulose or PVDF. Activate PVDF with methanol for 1 min and rinse with transfer buffer before preparing the stack. The time and voltage of transfer may require some optimization. We recommend following the manufacturer’s instructions. Transfer of proteins to the membrane can be checked using Ponceau S staining before the blocking step.
Prepare the stack as follows:
Figure 1. Example of prepared stack.
All lanes: beta Actin antibody - loading control (ab8227) at 1/5000 dilution
Lane 1: HeLa whole cell extract
Lane 2: Yeast cell extract
Lane 3: Mouse brain tissue lysate
Protocols are provided by Abcam “AS-IS” based on experimentation in Abcam’s labs using Abcam’s reagents and products; your results from using protocols outside of these conditions may vary.
The purpose of western blotting is to separate proteins on a gel according to the molecular weight. The proteins are then transferred onto a membrane where they can be detected using antibodies. Heat the samples and 95 degrees C for five to 10 minutes in a sample buffer containing a reducing agent such as beta mercaptoethanol. This results in linearized proteins with a negative charge proportional to their size.
Place a gel into the electrophoresis tank and ad in buffer, ensuring the tops of the wells are covered. Acrylamide percentage of the gel being used depends on the molecular weight of the target protein. Node a molecular weight market into the first lane then load the samples into adjacent wells. All the samples which contained equal amounts of protein. Once all the samples are loaded, ad running buffer, place the lid onto the electrophoresis tank. Turn on the power supply and set the voltage recommended by the manufacturer of the gels in the gel tank. You should be able to see bubbles rising through the tank. Run the gel until the die front has moved sufficiently down the gel.
The next stage is to transfer the proteins from the gel onto a membrane. Membranes are usually made from nitrocellulose or PVDF. Remove the gel from the tank and carefully release it from its plastic case. Cut up the wells and the gel foot and place the gel into transfer buffer. Prepare the transfer stack by sandwiching the membrane and gel between filter paper and sponges. The membrane should be traced to the positive electrode and the gel closest to the negative electrode. Use a small roller to remove any bubbles between the gel and the membrane. Cap the transfer case closed and submerge into a transfer tank containing transfer buffer. Add water to the outer chamber to keep the system cool and put on the lid. Turn on the power supply to begin protein transfer. Time and voltage require optimization, so check the manufacturer's instructions for guidance.
Now that the proteins have migrated from the gel onto the nitro cellulose membrane, the protein of interest can be detected as an antibody. The membrane can be removed from the cassette and the molecular weight market should now be visible. If required, the transfer of proteins can be confirmed by staining the membrane with [inaudible 00:04:40] solution. To prevent nonspecific binding of the antibody, the membrane needs to be blocked. Pour blocking buffer onto the membrane and agitate gently on a rocker. Typically, this is done using a solution of five percent milk or bovine serum albumin, BSA, for two hours at room temperature or overnight at four degrees. The time and type of blocking buffer should be optimized, so check the data sheet of the primary antibody you intend to use for details.
After the membrane is blocked, remove the blocking buffer and add the diluted primary antibody in the same solution. Incubate on the rocker as before. Typically primary antibody incubations are for one hour at room temperature or overnight at four degrees C. Antibody concentration and incubation time will need to be optimized. Refer to the antibody datasheet for guidance. Pour off the primary antibody and rinse the membrane twice in wash buffer. Follow with one 15 minute wash and three 10 minute washes on a rocker. The wash buffer is usually Trys buffered saline, TBS, or phosphate buffered, saline, PBS, with 0.1 percent tween 20.
Pour off the wash buffer and incubate the membrane in conjugating secondary antibody which has been diluted in blocking buffer. Usually this is done for one hour at room temperature, but antibody concentration and incubation time will need to be optimized. Pull off the secondary antibody and wash the membrane has shown previously.
There are several different systems for detection. If the secondary antibodies conjugate into an enzyme, incubate the membrane in the appropriate substrate before imaging. If the secondary antibodies are fluorescent counjugates then you can move directly onto the imaging step. Imaging can be carried out with x Ray film or with a digital imaging system. Place the membrane into an imaging tray. Place the imaging tray into imaging system. Exposure times will most likely need to be optimized in order to clearly detect the bands relating to the proteins of interest.