![]() HEK-293 cells were collected by trypsinization, washed and pelleted by centrifugation. CRL-1573, ATCC, Manassas, VA, USA), which are used commonly as transfection host cells, was tested. (B) Schematic view of Western blotting steps using single-sided incubation and conventional immersion incubation methods.Īs proof-of-principle, expression of endogenous beta-actin in human embryonic kidney 293 cells (HEK-293) (Catalog No. (A) Detailed description of single-sided incubation (left side of the Figure) and conventional immersion incubation (right side of the Figure). Single-sided antibody incubation and conventional immersion incubation The comparison of procedures of single-sided incubation and conventional immersion incubation is shown in Fig.1A. In this study, an economical, simple, straightforward, single-sided antibody incubation method for Western blotting is described, which generates significant savings on the use of antibodies. Although the unbound antibody can be reused between experiments, it is difficult to control the quantity and quality of reused antibody which may compromise the results. The procedure is much more complicated and it is especially useful for simultaneous protein detection of different antibodies when using limited samples. Microblots are now available which use just 100 µl or less of diluted anti sera ( Canzler et al., 2009a Canzler et al., 2009b). The reagent runs out very easily and the membrane may dry when applying reagent to one side of the membrane. However, using this method requires special bags and extra care to prevent folds in the membrane. Incubating membranes in heat sealed bags/sealed plastic bags or applying reagents directly to one side of the membrane has been used to reduce the cost of expensive reagents ( Dorri et al., 2009). Any method which uses the minimum amount of antibody without compromising the result is of obvious utility. Many antibodies are obtainable easily from commercial sources but remain relatively expensive and unaffordable for some laboratories. Specific and high-quality antibodies are essential for Western blotting as they are able to bind specifically to a target protein in a mixture of thousands of proteins on a standard Western blot. Western blotting is used widely in the fields of molecular biology, biochemistry, immunogenetics and other disciplines. The technique, allows determination of the approximate molecular weight of a protein and measurement of the relative amounts of the protein present in different samples ( Renart et al., 1979 Towbin et al., 1979). It uses gel electrophoresis to separate proteins, which are then transferred to a membrane where they are detected by probing with specific antibodies. Western blot (also known as protein immunoblot) is an analytical technique used for detection of specific proteins in a given sample of tissue homogenate or extract. ![]()
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