In Brief: An Intro to Western Blotting, and Your Questions Answered

The Origin and Methods of Western Blotting

What is Western Blotting?

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.

Why is it called “Western”?

“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”.

What are the steps of a Western blot procedure?

1. Lysate/Cell Preparation. The number one thing you can do for success in your Western blot is appropriate sample prep. You will never be able to visualize your proteins if they remain trapped in 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. Transfer. Getting the separated proteins out of the gel and bound to a membrane allows for easier detection. This is the procedure that I will highlight in this article.
4. Detection. This step involves incubating the transfer membrane in a solution containing an antibody to the protein of interest. When this antibody binds to the protein on the membrane, it can be detected with a chemiluminescent or fluorescent tagged secondary antibody allowing for visualization of the protein band.

Membrane Transfer Fundmentals

Why transfer the proteins to a membrane?

1. Ease of handling. Gels are fragile. We’ve all torn one. Membranes are hardier and are more easily manipulated.
2. Improved detection. Proteins are buried in the relatively thick gel. Getting them out of the gel and bound to the thin membrane allows for them to be more accessible to antibodies for detection.

Membranes? Tell me more.

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.

Pros:

1. Lower background than PVDF (in part due to a lower binding capacity).
2. Easier to block – less non-specific binding.
3. Does not need to be pre-wet with methanol.
4. Less expensive than PVDF.
5. Better for low MW proteins whose binding is enhanced since methanol (which shrinks membrane pore size) is in the transfer buffer.

Cons:

1. Lower binding capacity than PVDF.
2. Fragile – membrane can be easily chipped or cracked.
3. Unsupported nitrocellulose can’t stand up to stripping and re-probing.
4. Worse for high MW proteins – the methanol in the transfer buffer and the subsequent reduced pore size of the membrane can cause high MW proteins to precipitate.

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.

Pros:

1. Resistant to solvents – can be easily stripped and re-probed.
2. Higher binding capacity than nitrocellulose = higher sensitivity.
3. Stronger and more resilient material – easier to work with.

Cons:

1. Higher background – (due in part to the higher binding efficiency).
2. Needs to be activated with methanol.
3. Usually more expensive than nitrocellulose.

Pore Size Matters

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.

Transfer Mechanics

How do I transfer proteins from my gels to the membrane?

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.

Electroblotting Transfer Techniques: What’s the difference?

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:

Pros:

1. Most widely used transfer system.
2. More quantitative.
3. Very high protein transfer efficiency – 80-100%.

Cons:

1. Lengthy transfer times – 1 hour to overnight.
2. Lots of buffer, messy to assemble.
3. Complex setup – lots of pieces, can introduce error.

 

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.

Pros:

1. Short transfer times – 10 minutes or less.
2. Very little buffer, less waste.
3. Simpler, less messy setup.
4. High throughput, convenient.
5. Sandwich components are sold preassembled.

Cons:

1. Patchiness and unevenness can be more of a problem due to the fast speed.
2. Preassembled sandwiches are expensive.
3. Lower protein transfer efficiency – 60-80%.
4. Notorious for trapping bubbles, which cause spots on the membrane.

Essential Double-Check

How do I know if my protein has transferred?

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

1. Coomassie – incubate your gel in Coomassie blue and then destain to see if there is any protein left in the gel. It is likely that not all of the highest molecular weight proteins are out of the gel, but it will give you a good idea if the mid and low range proteins migrated out successfully.
2. Ponceau S – incubate your membrane in Ponceau S to visualize the banding pattern of the protein on your blot. This allows you to check for transfer efficiency as well as to highlight any problem areas on your gel.

Troubleshooting Your Transfer

Read about how to diagnose some common transfer issues here.

Western Blot Protocol

Read and download PhosphoSolutions’ Western Blot protocol here.

What makes us different?

1. If we attempt to make an antibody and it doesn’t work, we throw it away. We never sell an antibody that we can’t get to work in our hands.
2. Our commitment to strict validation standards ensures a clean signal in endogenous protein and verifies phosphospecificity for relevant products.
3. We are dedicated to reproducibility. Look for our “Pooled Serum” icon. Each antibody with this icon is purified from its own pool of serum to ensure lot-to-lot consistency.
4. We guarantee our antibodies. If you can’t get the antibody to work in your application, we will provide a 100% refund.
5. When making a custom antibody, we provide personalized customer support from start to finish, from peptide design, to purification, to characterization.