Western Blot is a technique commonly used in scientific research laboratories. By means of this technique, the different proteins present in a sample are separated according to their molecular weight by gel electrophoresis, and are subsequently transferred to a membrane to proceed with their identification using specific antibodies.
Although the foundation of the technique remains, over the years new detection methods have been developed in order to obtain more accurate results and also allow a quantitative analysis of proteins.
In this post we bring you a summary of the main detection methods for Western Blot with the advantages and disadvantages of each of them.
DETECTION METHODS FOR WESTERN BLOT
1.- RADIOACTIVE DETECTION METHODS
This type of detection was one of the first methods used to reveal the results of the Western Blot, by labeling the antibodies with radioactive conjugates.
The main advantage of this method lies in its sensitivity, but it has the great drawback that when using radioactive materials there is a risk to the researcher’s health and safety. Furthermore, it is a high-cost technique and its execution is time consuming.
Radioactive detection is not currently among the Western Blot detection methods of choice. In fact, its use is discouraged.
2.- ENZYMATIC DETECTION METHODS
These methods are based on the use of secondary antibodies conjugated to an enzyme that catalyzes a reaction with a specific substrate.
Within this category, detection can be carried out by means of two types of enzymatic reactions:
2.1 COLORIMETRIC DETECTION
In this case, the enzyme bound to the secondary antibody triggers a reaction with the substrate giving rise to a colored precipitate that can be visually identified.
The advantages of this method lie in its speed, simplicity and low economic cost, in addition to not requiring any special equipment. Its drawback is its low sensitivity (in the order of picograms).
This method is usually used when it is necessary to quickly and easily analyze the presence or absence of a certain protein.
2.2 DETECTION BY CHEMIOLUMINISCENCE
In chemiluminescence assays, the enzyme bound to the secondary antibody triggers a reaction with a luminescent substrate generating light.
In this case, the great advantage is the high sensitivity provided by this method (in the order of femtograms), allowing proteins with very low levels of expression to be identified. As a drawback, note that it requires the use of specialized equipment to read the results.
3.- FLUORESCENT DETECTION METHODS
This type of detection is based on the use of secondary antibodies conjugated to fluorophores that produce signal by themselves, without the need to add any additional substrate.
Among the advantages, it should be noted that the signal is more stable than that produced by enzymatic detection methods and, above all, that the possibility of using fluorophorized antibodies with different emission wavelengths on the same Western Blot membrane allows multiplexing the experiments. It should also be noted that this method also allows quantifying the protein present in the sample.
As disadvantages, a lower sensitivity than chemiluminescence detection, and the need to use specialized equipment.
Fluorescence is among the most widely used Western Blot detection methods today.