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Western blot workflow. The western blot (sometimes called the protein immunoblot), or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. [1]
BCA protein assay in a 96 well plate. The bicinchoninic acid assay (BCA assay), also known as the Smith assay, after its inventor, Paul K. Smith at the Pierce Chemical Company, [1] now part of Thermo Fisher Scientific, is a biochemical assay for determining the total concentration of protein in a solution (0.5 μg/mL to 1.5 mg/mL), similar to Lowry protein assay, Bradford protein assay or ...
Fluorescent stains and stain-free gels require special equipment to visualize the proteins on the gel/blot. [5] Stains may not cover the blot evenly; more stain might collect towards the edges of the blot than in the center. Non-uniformity in the image can result in inaccurate normalization. [1]
This reaction is rapid and stoichiometric, with the addition of one mole of thiol releasing one mole of TNB. The TNB 2− is quantified in a spectrophotometer by measuring the absorbance of visible light at 412 nm, using an extinction coefficient of 14,150 M −1 cm −1 for dilute buffer solutions, [4] [5] and a coefficient of 13,700 M −1 cm −1 for high salt concentrations, such as 6 M ...
Other interference may come from the buffer used when preparing the protein sample. A high concentration of buffer will cause an overestimated protein concentration due to depletion of free protons from the solution by conjugate base from the buffer. This will not be a problem if a low concentration of protein (subsequently the buffer) is used. [6]
Similar to a western blot, the far-western blot uses protein–protein interactions to detect the presence of a specific protein immobilized on a blotting matrix. Antibodies are then used to detect the presence of the protein–protein complex, making the Far-Western blot a specific case of the Western blot.
In a generally healthy low-risk population, indeterminate results on western blot occur on the order of 1 in 5,000 patients. [15] However, for those individuals who have had high-risk exposures to individuals where HIV-2 is most prevalent, Western Africa, an inconclusive western blot test may prove infection with HIV-2. [16]
Label-free quantification is a method in mass spectrometry that aims to determine the relative amount of proteins in two or more biological samples. Unlike other methods for protein quantification , label-free quantification does not use a stable isotope containing compound to chemically bind to and thus label the protein.