Western blot, an analytical technique used to pinpoint a specific protein in a given sample, employs the ability of an enzyme or fluorescence-labeled primary antibody to bind to its specific antigen. It is a three-step process beginning with gel electrophoresis, followed by membrane blotting and probing with antibodies. Protein detection may be direct or indirect, with the latter using a labeled secondary antibody directed against the primary. Though accepted as a routine protein analysis technique, western blot has limitations as well as benefits.
One of the biggest arguments in favor of western blot is its sensitivity. Because of its ability to detect as little as 0.1 nanograms of protein in a sample, the technique can theoretically serve as an effective early diagnostic tool, sensing even the slightest immunogenic response from a virus or bacteria in a patient sample. An indirect western blot further builds on this sensitivity from the secondary antibody’s ability to amplify the intensity of the signal detected by the imaging system. Greater sensitivity means that fewer antibodies are needed for testing, which cuts down laboratory costs significantly.
The western blot technique owes its specificity to two big contributing factors. First, gel electrophoresis sorts a sample into proteins of different size, charge, and conformation. This process in itself is a huge step towards detection, as bands formed in the gel already give clues about the size of the protein or polypeptide of interest. The specificity of the antibody-antigen interaction serves as the second big factor. Because specific antibodies show affinity for specific proteins, the process can selectively detect a target protein even in a mixture of 300,000 different proteins.
Disadvantage: Prone to False or Subjective Results
In spite of its sensitivity and specificity, a western blot can still produce erroneous results. A false-positive results when an antibody reacts with a non-intended protein, which is what frequently happens when a patient being tested for HIV happens to have tuberculosis or a number of parasitic infections. A false-negative, on the other hand, can easily result if larger proteins are not given sufficient time to transfer properly to the membrane. Improper blotting and processing often produce skewed, faded, or even multiple bands, making test results subject to the interpretation of the technician.
Disadvantage: High Cost and Technical Demand
The cost of a western blot is a composite of the large individual expenditures for tagged antibodies, skilled analysts and laboratory equipment. A delicate process, western blotting requires precision in every step for proper identification of a sample’s constituents. A minor error in reagent concentration or in incubation period can be disastrous for the entire process. Finally, the equipment required for detection and imaging – chemiluminescent, fluorescent, radioactive, or laser detection systems – can be too expensive for the average microbiology unit.