In Brief: An Intro to Western Blotting, and Your Questions Answered
The basic concepts and history of Western Blotting are often jumped over when diving straight into the technique. Here is your primer.
by Amy Archuleta
by Amy Archuleta
The Western Blot (or immunoblot) technique uses antibodies to detect protein targets that have been bound to a membrane. It was introduced in 1979 by Harry Towbin’s research lab in Switzerland.
“Western” is a play on words based on a similar technique. Edwin Southern published a DNA detection technique in 1975 from his lab in England. It involved transferring DNA to a membrane and was dubbed a “Southern blot” in honor of its inventor’s name. Two years later, researchers at Stanford developed a similar RNA detection technique that was dubbed “Northern blot”. Two years after that, when Towbin transferred proteins to a membrane, it eventually became known as a “Western blot”.
Nitrocellulose and PVDF (polyvinylidene difluoride) are the membranes of choice for most Western blotting applications. Both membranes are microporous substrates that bind proteins to their surface through hydrophobic interactions. Here are the basic differences between the two:
Nitrocellulose. One of the first membranes used in Western blotting. It can bind protein at a capacity of 80–100 µg/cm2.
PVDF (polyvinylidene difluoride). PVDF is a popular alternative to nitrocellulose due to its high binding and strength. Its binding capacity is 170-200 µg/cm2.
Regardless of which membrane you use, you also need to consider pore size. Both types of membrane are microporous. The size of the pore determines the size of the protein that can bind without passing through. Membranes are available in different pore sizes, most commonly 0.2 µm and 0.45 µm. For most proteins, the 0.45 µm size works well. For low MW proteins, <20 kDa, it is a good idea to use a membrane with a smaller pore size to keep your protein of interest from passing through the pores.
Most scientists use electroblotting to transfer their proteins from gels to membrane. A “transfer sandwich” of filter paper – gel – membrane – filter paper is placed between two electrodes. (In wet transfer systems, there is a sponge on either side of the sandwich.) The negatively charged proteins in the gel are pulled in an electric current toward the positively charged anode and into the membrane. Since the gel and membrane are sandwiched tightly during the procedure, the proteins maintain the separation they achieved during the SDS-PAGE electrophoresis. Prior to electroblotting, protein transfer could be performed through capillary transfer. This involves the same sandwich of filter paper, membrane, and gel, but relies on capillary action to pull transfer buffer from a lower reservoir, through the gel/membrane, and to the filter paper on the top, bringing the proteins with it and depositing them on the membrane. This method is not frequently used due to the lengthy procedure time, but it is decidedly less expensive than electroblotting because no fancy apparatus is needed.
There are two primary electroblotting techniques: wet tank transfer or semi-dry transfer.
Wet-tank transfer – the “sandwich” described above – with sponges – is placed in between two electrodes and submerged vertically in a chamber containing transfer buffer. Some common tank transfer systems are shown here:
Semi-dry transfer – the sandwich described above – without sponges – is placed horizontally in between two plate electrodes. The filter paper is wetted with transfer buffer, but there is no buffer reservoir or submerging of the transfer sandwich.
Before starting your antibody incubation step, there are two staining procedures we recommend to make sure your transfer was successful.
Read about how to diagnose some common transfer issues here.
Read and download PhosphoSolutions’ Western Blot protocol here.