The microscale Western blot uses capillary gel electrophoresis to move a small sample of liquid through a gel-filled tube. It can be used as a diagnostic test to detect and measure the concentration of specific proteins, antibodies or antigens in small samples of blood and other bodily fluids.
The microscale Western blot can also be used to purify, characterize, and quantify antigens for use in other testing and research. Medical applications include testing for HIV-AIDS, Hepatitis, feline immunodeficiency virus (FIV), Lyme syphilis, and Bovine spongiform encephalopathy.
Western blotting is widely used for selective protein identification based on size and antibody binding.
The microscale Western blot uses an electrical field to move the liquid sample through a thin capillary tube filled with a polymer gel. The gel is able to separate proteins from the sample and trap them. Because larger molecules travel through the gel at different speeds than smaller molecules, the proteins become sorted by size.
After the sample passes through the tube the trapped proteins are washed out and directly transferred to blotting paper made from a polyvinylidene fluoride (PVDF) membrane that has been treated with substances which bind to the proteins. When the proteins react with these substances, they are revealed on the paper as dark bands of varying widths, according to molecule size. This process is a type of affinity chromatography, making use of the attractions that occur between certain substances, such as between antigen/antibody pairs and producing a visual output.
Microscale Western Blot Capillary Gel Electrophoresis - Advatanges Over Conventional Western Blotting
One of the main advantages microscale Western blot has over conventional slab Western blotting is that it eliminates electro-blotting needed to deposit the proteins from the gel slab onto the blotting paper. Electro-blotting adds time and expense, and it requires additional equipment. The microscale Western blot deposits the proteins directly on the paper.
Slab Western blotting requires larger volumes of both sample and reagent, and the agarose gel used is difficult to use with large proteins and is not effective with low-concentration sample.
Microscale Western blotting tests take less time to prepare and conduct than the conventional Western blot. A test of a low-concentration (micrograms per milliliter) liquid sample for presence of a protein was completed in about an hour, compared to typical times of 4–24 hours. The test also sensitivity in the nanogram range.
- Diagnostic test for HIV-AIDS by detecting anti-HIV antibody
- Diagnostic test for Bovine spongiform encephalopathy
- Confirm Feline immunodeficiency virus (FIV) status in cats
- Confirmatory test for Hepatitis infection
- Lyme disease testing
- as the confirmatory test for syphilis
- to purify, characterize, and quantify antigens
- Tests can be completed in about 1 hour, compared to 4-24 hours with conventional test
- Effective for use with low-concentration samples
- Effective for large proteins
Eliminates electro-blotting step from conventional slab Western blotting
Requires fewer pieces of equipment, which saves room and reduces costs
Works with smaller samples
Uses less reagent
Requires less time to prepare and conduct
Less manually intensive