![]() ![]() ![]() Unfortunately, a limitation of this class of markers is that they must be used under non-denaturing conditions, which may lead to inaccurate determination of molecular weight. Immunoglobulin (Ig)-binding domain fusion proteins have been developed as auto-color-development molecular weight markers, such as the EasySee Western marker (Spark Biologicals Technology) which contains IgG-binding domains of protein A and protein G. Bischof and colleagues developed a heme-based ladder which possess peroxidase activity that can be revealed by substrate treatment. For example, Chang and colleagues developed a green fluorescent protein (GFP)-based protein ladder which can be monitored on SDS-PAGE gel under ultraviolet (UV) illumination. To overcome this problem, a number of dye-free and automatically color developed molecular weight markers have been developed by fusion of the functional domains of specific proteins. However, the conjugation of dye molecules to protein markers may altered their electromobility and impede precise molecular weight determinations. Currently, dye-conjugated molecular weight protein markers are commonly used in protein analysis. Molecular weight markers that can be auto-detected by secondary antibodies and tolerate denaturing conditions can offer great convenience for protein analysis by Western Blotting. This accurate, regular and auto-detected M&R LE protein marker may provide a simple, efficient and economical tool for protein analysis. Linear regression analysis of the M&R LE protein marker plotted as gel mobility versus the log of the marker molecular weights revealed good linearity with a correlation coefficient R 2 value of 0.9965, indicating that the M&R LE protein marker displays high accuracy for determining protein molecular weights. The M&R LE protein marker can be auto-detected by anti-mouse and anti-rabbit IgG secondary antibodies in standard immunoblots. We fused the M2 and R2 epitopes (M&R LE) and incorporated the polypeptide in a range of 15–120 kDa auto-detecting markers (M&R LE protein marker). Western blot analysis indicated that M2 and R2 linear epitopes are effectively recognized by anti-mouse and anti-rabbit secondary antibodies, respectively. We selected three mouse (M1, M2 and M3) linear IgG1 and three rabbit (R1, R2 and R3) linear IgG heavy chain epitope candidates based on their respective crystal structures. These markers can be directly recognized and stained by a wide range of anti-mouse and anti-rabbit secondary antibodies. Here, we describe M&R LE protein markers which contain linear epitopes derived from the heavy chain constant regions of mouse and rabbit immunoglobulin G (IgG Fc LE). Molecular weight markers that can tolerate denaturing conditions and be auto-detected by secondary antibodies offer great efficacy and convenience for Western Blotting. ![]()
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