What are the differences between Western Blot and ELISA assays?
What is Western Blot?
Western blot is an analytical technique used to detect specific proteins which have been separated by gel electrophoresis according to size. The separation technique relies on an electrical current being transferred through a buffer solution enclosed in an electrophoresis chamber by a connected electrode (see Figure 1). Proteins are then transferred via the wet-transfer method from the gel to a membrane. This is usually made from nitrocellulose or PVDF (see Figure 2). The membrane is then probed with specific antibodies which target the protein marker. Since biological reagents are colourless, these antibodies are radioactively labelled with fluorescent proteins which tend to be green or red. This labelling allows us to see the size of the band (kDa) using luminescent imaging of the membrane, where band density can be measured (see Figure 3).
Fig 1: shows the set up of an electrophoresis chamber
Fig 2: shows the transfer process during Western Blotting
Fig 3: shows the image produced by Western blotting, with banding of the sample protein.
What is ELISA?
ELISA stands for enzyme linked immunosorbent assay and has many different applications such as quality control in industry. As well as a diagnostic tool in pathology and medicine. Antigens from a test sample are greatly diluted and attached to a surface. This surface tends to be a multiwell plate in which a pre-determined layout of sample is organised.
A specific antibody is added which binds to the sample antigen. The antibody used in this step is linked to an enzyme, so that in the final stage the enzyme’s substrate can be added. A compilation of these reactions produces a detectable signal such as a colour change, resulting from the binding of the substrate to its complimentary enzyme to form an enzyme-substrate complex (see Figure 4). Samples which can be tested include viral particles, food antigens or proteins. The multi-well plate is then read by a plate reader to give a qualitative, quantitative or semi-quantitative result. This information can be used accordingly.
Fig 4: shows the ELISA binding reactions