The sample material undergoes protein denaturation, followed by gel electrophoresis. Next, the electrophoresis membrane is washed in a solution containing the specific antibody. The excess antibody is then washed off, and a secondary antibody that reacts with the first antibody is added. Through various methods western blotting procedure pdf as staining, immunofluorescence, and radioactivity, the secondary antibody can then allow visualisation of the protein.
The term “western blot” was given to the technique by W. It is used as a general method to specifically prove the existence of a single protein in a mixture. A semi-quantitative estimation of a protein can be derived from the size and color intensity of a protein band on the blot membrane. It is also used in medical diagnostics, e. The stained bands then indicate the proteins to which the patient’s serum contains antibody. Each is prohibited under WADA’s List of Prohibited Substances and Methods. The western blot technique was used during the 2014 FIFA World Cup in the anti-doping campaign for that event.
In total, over 1000 samples were collected and analyzed by Reichel, et al. WADA accredited Laboratory of Lausanne, Switzerland. Recent research utilizing the western blot technique showed an improved detection of EPO in blood and urine based on novel Velum SAR precast horizontal gels optimized for routine analysis. With the adoption of the horizontal SAR-PAGE in combination with the precast film-supported Velum SAR gels the discriminatory capacity of micro-dose application of rEPO was significantly enhanced. Prior to electrophoresis, protein samples are often boiled to denature the proteins present.
Lastly, the membrane will be stained with a secondary antibody that recognizes the first antibody staining, which can then be used for detection by a variety of methods. Samples can be taken from whole tissue or from cell culture. Cells may also be broken open by one of the above mechanical methods. However, virus or environmental samples can be the source of protein and thus western blotting is not restricted to cellular studies only. The nature of the separation depends on the treatment of the sample and the nature of the gel.
This is a very useful way to identify a protein. The concentration of acrylamide determines the resolution of the gel – the greater the acrylamide concentration, the better the resolution of lower molecular weight proteins. The lower the acrylamide concentration, the better the resolution of higher molecular weight proteins. Proteins travel only in one dimension along the gel for most blots. The proteins move from within the gel onto the membrane while maintaining the organization they had within the gel. An older method of transfer involves placing a membrane on top of the gel, and a stack of filter papers on top of that. Protein binding is based upon hydrophobic interactions, as well as charged interactions between the membrane and protein.
Nitrocellulose membranes are cheaper than PVDF, but are far more fragile and do not stand up well to repeated probings. This step visualizes the total protein that has been successfully transferred to the membrane. Total protein staining allows the user to check the uniformity of protein transfer and to perform subsequent normalization of the target protein with the actual protein amount per lane. Normalization with the so-called “loading control” has based on immunostaining of housekeeping proteins in the classical procedure, but is heading toward total protein staining recently, due to multiple benefits. In order to avoid noise of signal, total protein staining should be performed before blocking of the membrane.