Blotting is mostly used in the molecular biology for identifying nucleic acids and proteins and is used for a number of other diagnostic purposes. Western blot is one of the most important analytical techniques, which is used for pinpointing a particular protein within a sample. This technique requires assistance from an enzyme, which is capable of binding with a specific antigen. It is true that this process is accepted as a great technique of protein analysis, but there are certain benefits and limitations that you should have a proper knowledge about.
What Is Western Blotting
Western Blotting has three steps involved, which include the gel electrophoresis, the membrane blotting, and finally, probing with the antibodies. The detection of the protein can be either direct or even indirect. The indirect procedure makes use of a secondary antibody, which is directed towards the primary antibody. To understand the procedure of Western Blotting, you can visit https://www.mybiosource.com/.
Listed below are few of the advantages that are associated with Western Blotting.
Specificity– This technique is specific and there are two factors behind this. The process of gel electrophoresis helps in sorting the sample into the proteins of various sizes, conformation, and charges. This process is considered to be one of the most important steps towards detecting, as it aids in detecting the size of the polypeptide or the protein.
The second factor is the specificity of antigen-antibody interaction. A particular antibody will show an affinity for a specific protein, the process helps in successfully detecting the target protein.
Sensitivity– The process is capable of determining even 0.1 nanograms of protein, it is considered to be one of the most effective and convenient diagnostic tools. The process is capable of sensing immunogenic responses from a bacterium or a virus in no time.
This is why the biggest advantage of the Western Blotting process is undoubtedly its sensitivity. A good sensitivity implies that few antibodies are required for the testing process, which plays an important role in cutting down the laboratory charges.
Given below are few of the disadvantages that are associated with Western Blotting.
Prone to Subjective and False Results– Despite the specificity and the sensitivity, there are chances that it can produce results that are erroneous. An incorrect positive may result when antibodies react with the non-intended protein.
This happens normally when a person who is supposed to be tested for the HIV virus has other parasitic infections or tuberculosis. Incorrect negatives result when the large proteins are not provided ample time for being transferred into the membrane.
Technical Demand and High Cost– Western Blotting is extremely costly because it involves expenditures for skilled analysts, antibodies, and laboratory equipment. It also requires the laser detection units, radioactive units, chemiluminescent units, for the detection and image purposes, which can be extremely expensive.
Though Western Blotting is costly and can lead to incorrect results at certain times, it is suggested by a number of doctors for detecting a positive or a negative condition in a person.