The Basics of Western Blotting

Our sister company, PhosphoSolutions, recently shared a blog post called "The Very Basics of Western Blotting" contributed by our own Amy Archuleta!

This is an abbreviated article.

Read the full-length piece in their website’s Education section. Regardless of whether you perform Western blots frequently in your lab or are new to the technique, here is a reminder of (or an intro to) the fundamentals behind the box.

Western Blotting is a protein detection technique.

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. It was the third technique developed in membrane transfer, after “Southern blotting” (for DNA) and “Northern blotting” (for RNA).

There are 4 basic steps in the Western Blot procedure.

1. Lysate/Cell Preparation. To be successful, you must get your proteins out of your tissue or cells. You can read about our recommended lysis buffer here. You can purchase our lysis buffer here. Read about lysing your samples here. Read our lysate preparation protocol here.
2. SDS-PAGE. Separation of the proteins in your lysate by molecular weight is done through electrophoresis. Read all about the technique here.
3. Getting the separated proteins out of the gel and bound to a membrane allows for easier detection.
4. This step involves incubating the transfer membrane in a solution containing an antibody to the protein of interest, and using a fluorescent tagged secondary antibody for visualization.

Transferring to a membrane makes antibody detection easier, but you need to consider which membrane to choose.

The biggest advantages to transferring to a membrane are that membranes are easier to handle than gels and they allow for easier detection by getting the proteins out of the gel onto a thinner substrate. 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. Nitrocellulose is cheaper and has lower background but is fragile and has a lower binding capacity than PVDF. PVDF has a higher binding capacity and is stronger but needs to be activated with methanol and is more expensive. Click on the full-length article for a more in-depth comparison of these two membranes. The pore size of the membrane is also important. 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, but for lower MW proteins, you should consider 0.2 μm.

Most scientists use an electric current to transfer their proteins from gels to membrane.

This is known as electroblotting. 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 a 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. 

There are two primary electroblotting techniques: wet tank transfer or semi-dry transfer.

1. Wet-tank transfer – the “sandwich” described above – with sponges – is placed in between two plate electrodes and submerged vertically in a chamber containing transfer buffer. This technique is the most widely used and has high efficiency but is messy and time consuming. Some common tank transfer systems are shown here:
2. 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. Semi-dry transfers are quicker and less messy but can be expensive, uneven and less efficient.

   Always check to make sure your protein has transferred.

   Before starting your antibody incubation step, there are two staining procedures we recommend to make sure your transfer was successful.

   1. 1. 1. Coomassie – incubate your gel in Coomassie blue and then destain to see if there is any protein left in the gel.
         2. Ponceau S - incubate your membrane in Ponceau S to visualize the banding pattern of the protein on your blot.

   
   Troubleshooting your transfer:
   Read about how to diagnose some common transfer issues here.
   
   Read the full-length article with additional details here.
   Read and download our Western Blot Protocol here.