BioTechniques on Western Blotting: History, Procedure, Analysis, Troubleshooting, and Purpose

An introduction to how and why researchers use western blotting to identify the presence and absence of proteins in samples.

Researchers often need to determine the presence or absence of a protein in a sample - and maybe the size of the protein too. Whether they're detecting or investigating a disease, determining the success or failure of genetic experiments, or identifying potentially allergenic proteins in food samples, researchers need to distinguish their target proteins from a background of many other proteins - some of which may share the target protein's properties or be of a similar size.

This is where the western blot comes in. Western blotting is an ideal technique for identifying proteins in these kinds of situations. Scientists and lab workers who are looking to keep up to date with updates in the technique's applications and troubleshooting solutions follow the life sciences journal BioTechniques. This peer-reviewed, open-access journal publishes articles that explain what western blotting is, how scientists perform the technique, what western blots reveal, and how they differ from similar techniques.

Where Did Western Blotting Come From?

Western blotting (or 'protein blotting' or 'immunoblotting') is an antibody-based technique that scientists use to detect the presence, size, and abundance of proteins in a sample. A postdoctoral fellow Harry Towbin and his colleagues developed the technique in 1971, and the postdoctoral researcher W Neal Burnette developed the technique further in 1981. Once finalized, the technique was named the western blot, a play on the similar Southern blot.

What Is Western Blotting?

Western blotting involves separating proteins in an aqueous sample through electrophoresis. Researchers then transfer the proteins to a membrane (the blot) and probe the sample using target-specific, detectable antibodies. They then compare the size and abundance of the bound proteins against known standards or controls.

How Does Western Blotting Work?

1.      Separating the Proteins

Researchers separate the proteins in a sample through protein electrophoresis, which separates proteins based on their electric charge, isoelectric point, molecular weight, or a combination of these. Researchers choose the separation method for the process depending on the aim of the analysis.

If a researcher needs a clean image, they must centrifuge the sample to remove any solids and load only the soluble fraction. If the protein of interest is in the insoluble fraction (like cell membrane-bound proteins), researchers should use pretreatment methods to liberate and solubilize the protein first. This is because solids impair the running of the gel, which means the protein of interest may stay in the stacking gel.

Researchers should also load appropriate control samples and size market ladders so they can interpret the final blot.

2.      Transferring/Blotting the Proteins

Researchers must transfer the proteins from the gel to an appropriate membrane, like a nitrocellulose or polyvinylidene difluoride (PVDF) membrane to enable antibody probing. They might employ one of many transfer techniques, such as vacuum blotting, diffusion transfer, capillary transfer, or electroblotting (also known as 'electroelution' or 'electrophoretic transfer'). Electroblotting is the most common technique because of its speed and efficiency benefits. The process involves applying an electrical current to a gel-membrane sandwich. This carries proteins from the gel to the membrane.

3.      Blocking the Binding Sites

After transferring the proteins from gel to membrane, researchers must block any remaining binding sites. This prevents non-specific binding of the assay detection antibodies. Researchers can block binding sites by incubating the membrane with a proteinaceous liquid like milk or serum.

4.      Washing the Blot

Once they've blocked the remaining binding sites, researchers must wash the membrane between each of the remaining steps to eliminate any excess or unbound reagents. Inadequate washing can lead to low-quality or patchy blots and high background. That said, over washing can diminish the target signal. Therefore, it's important to optimize the number and duration of wash steps. Researchers should cover the membrane with an appropriate buffer and gently wash the membrane to avoid damaging it.

5.      Applying Primary and Secondary Antibodies

Although researchers may use direct detection (a single detectable antibody that recognizes the target protein), they're more likely to apply an indirect method in which they use a primary antibody to probe the membrane and bind the target protein (if it's present). They then apply a secondary antibody (also detectable) to bind the primary antibody. It's essential to select an appropriate antibody (and concentration of this antibody) to achieve a good-quality blot.

When using an indirect detection assay, researchers need to wash excess unbound primary antibody off the membrane and then apply a secondary antibody. They should use a secondary antibody that is specific to the series of the primary antibody and possesses the required conjugate for the detection method.

6.  Analyzing the Western Blot

Researchers can employ one of various methods to detect and visualize western blots, e.g. chemiluminescent, colorimetric, fluorescent, and radioactive detection methods. Chemiluminescent and colorimetric detection techniques are sensitive and need the conjugation of an enzyme (like horseradish peroxidase (HRP) or alkaline phosphatase (AP)) to the detection antibody. Researchers add a substrate to the membrane, which the conjugated enzyme acts on, triggering a chemical change. Meanwhile, fluorescent detection involves conjugating the detection antibody with a fluorophore instead of an enzyme.

Radioactive detection requires researchers to conjugate a radioisotope to the detection antibody and detect the emitted radiation on x-ray film. However, researchers must follow precautions to protect themselves against radiation. Plus, this detection method is costly and has a limited shelf life because of radioactive decay. Therefore, the other three methods are more commonly used.

7.  Stripping the Buffer

Stripping is the process of removing primary and secondary antibodies from the membrane. Researchers may need to do this if they are investigating more than one target protein on an individual blot. However, stripping can remove some proteins from the membrane. Therefore, researchers shouldn't make quantitative comparisons before or after stripping and shouldn't check for the absence of a protein on a stripped membrane.

Researchers who are stripping a blot may use PVDF membranes, which are better at retaining proteins than nitrocellulose. Researchers should use mild, low pH stripping buffers that keep sample loss at a minimum. They should only try more stringent stripping through detergent and heat-based methods if this doesn't work. In this case, it's also a good idea to use chemiluminescent detection as colorimetric detection leaves a permanent stain.

How Do Researchers Measure Western Blot Results?

Researchers can use x-ray film to obtain results from a western blot experiment. They may need to expose several films so they can optimize exposure and developing time. If they don't leave the film for long enough, the bands may be faint or not visible at all. But if they leave the film for too long, the background signal may be too strong, which can cause the bands to merge. This can make the blot dark and hard to interpret. Controls or size markers that aren't balanced in concentration with the target can also pose difficulties. Once researchers have obtained good-quality results from an x-ray film, they can digitize the films for software analysis.

Western blotting x-ray film detection methods are qualitative or semi-quantitative. Researchers can compare bands in sample lanes against controls and size markers to identify the presence or the absence of a target protein in a sample. However, if researchers require quantitation, they may use digital imaging with CCD camera-based devices instead of x-ray films. These devices offer a larger dynamic range, higher resolution, and greater sensitivity. Researchers also may adjust their exposure times without repeating time-consuming x-ray film exposures and developments.

How Do Researchers Troubleshoot Western Blots?

Unfortunately, there are many opportunities during the western blotting process for things to go wrong. Examples of western blotting faults include:

• The insufficient separation or poor positioning of protein bands on the gel (and then the blot). Researchers should optimize the protein gel percentage and run time to suit the target protein.
• Nothing on the blot (or even the ladder). Researchers should make sure that the blotting kit has been assembled in the correct order and that the current is running in the correct direction. Otherwise, the sample will blot into the buffer.
• The blot appears patchy or messy. Researchers should make sure that there are no bubbles between the gel and the membrane when setting up the transfer. They should ensure that the membrane is fully submerged and carefully mixed during incubation. They should also use a fresh blocking reagent.
• A high background. This often occurs when blocking or washing is inadequate.
• Results of control samples that are inconsistent with the expected outcome. Researchers should optimize the choice and concentration of the antibody to reduce the likelihood of off-target binding.
• Bands that are too light or dark to see clearly on the final blot. Researchers should optimize the developing times in the detection phase to improve signal strength. Bands that are too light could alternatively suggest over washing. Researchers can optimize the type and concentration of the antibody to help here too.

What Is the Purpose of Western Blotting?

Western blotting enables scientists to identify the presence or absence of a target protein in a sample. More specifically, the western blot enables scientists to evaluate protein-DNA interactions, protein-protein interactions, post-translational modifications (PTMs), protein isoform detection, antibody characterization, epitope mapping, and subcellular protein localization.

As a result, common applications of western blotting include determining whether a protein is expressed, detecting tagged proteins, mapping changes over time or between groups, identifying biomarkers of non-infectious diseases, and diagnosing infectious diseases like Lyme disease, HIV, bovine spongiform encephalopathy (BSE), and aspergillosis.

About BioTechniques

BioTechniques publishes the latest information in life sciences lab techniques and methodologies, exploring the reproducibility and efficacy of these instead of focusing on treatments. Scientists, lab workers, and other industry experts from a variety of disciplines like chemistry, physics, computer science, and plant and agricultural science keep up with both BioTechniques and the journal's multimedia website, making the most not only of webinars, videos, interviews, industry articles, podcasts, and eBooks.