Abstract
Molecular weight markers that can tolerate denaturing conditions and be auto-detected by secondary antibodies offer great efficacy and convenience for Western Blotting. 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). These markers can be directly recognized and stained by a wide range of anti-mouse and anti-rabbit secondary antibodies. 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. Western blot analysis indicated that M2 and R2 linear epitopes are effectively recognized by anti-mouse and anti-rabbit secondary antibodies, respectively. 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). The M&R LE protein marker can be auto-detected by anti-mouse and anti-rabbit IgG secondary antibodies in standard immunoblots. 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 R2 value of 0.9965, indicating that the M&R LE protein marker displays high accuracy for determining protein molecular weights. This accurate, regular and auto-detected M&R LE protein marker may provide a simple, efficient and economical tool for protein analysis.
Highlights
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
To develop a denaturable molecular weight marker that can be directly recognized by secondary antibodies, we designed marker proteins with linear epitopes derived from the Fc region of mouse IgG1 and rabbit immunoglobulin G (IgG) antibodies (IgG Fc LE) for binding by commercial secondary antibodies
IgG Fc LE candidate sequences from mouse (M1, M2 and M3) primary IgG1 and rabbit (R1, R2 and R3) IgG antibodies were predicted based on their crystal structures by ABCpred, BepiPred 1.0 Server and Cancer Vaccine Center (CVC) bioinformatics software (Table 1 and Fig 2)
Summary
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. The conjugation of dye molecules to protein markers may altered their electromobility and impede precise molecular weight determinations [3]. 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. 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 [6]. Development of an autodetected, denaturable, precise and widely applicable molecular weight marker is desirable
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