Fig 1.
Construction of M&R LE protein marker for Western blotting.
We fused mouse and rabbit linear eptitopes (M&R LE) to generate a 15 kDa leader peptide which can be recognized by anti-mouse or anti-rabbit IgG secondary antibodies. The M&R LE molecular weight ladder was constructed by fusing a leader peptide with the E. coli outer membrane protein intimin domain 2 (D2), maltose-binding protein (MBP) or E. coli transcription factor (Nus) polypeptides. The M&R LE protein marker can be directly visualized on Western blotting by staining with anti-mouse or anti-rabbit IgG secondary antibodies. SP, signal peptide; M2R2, M&R epitope; RE, restriction enzyme sites; MAS, MW adjusting sequence; H, 6X His tag.
Table 1.
The amino acid sequences of linear epitope candidates of mouse IgG1 and rabbit IgG.
Fig 2.
Prediction of linear epitope in mouse and rabbit antibody IgG Fc.
To select linear epitopes, we predicted the crystal structures of mouse IgG1 and rabbit IgG Fc by ABCpred, BepiPred 1.0 Server and Cancer Vaccine Center (CVC) bioinformatics software. The 3D structures of mouse IgG1 and rabbit IgG Fc are displayed by PyMol software. (A) Three linear epitope candidates for mouse IgG1 Fc are shown as M1 (red), M2 (blue) and M3 (yellow). (B) Three linear epitope candidates for rabbit IgG Fc are shown as R1 (red), R2 (blue) and R3 (yellow).
Fig 3.
Expression and recognition of M&R LE leader peptides by mouse and rabbit secondary antibodies.
The LE candidates (M1, M2, M3, R1, R2 and R3) and M&R LE leader peptide (M2R2) were separated by a 12.5% SDS-PAGE and transfered onto NC membrane. The expression of polypeptides was detected by staining with (A) anti-His tag antibody and standard Western Blotting. The recognition of LE candidates and M&R LE leader peptides on immunoblots by (B) HRP-conjugated anti-mouse or (C) HRP-conjugated anti-rabbit IgG Fc secondary antibodies. The chemiluminescence was captured by a UVP BioImaging system. The relative molecular weight (kDa) of commercial prestained markers is indicated on the left.
Fig 4.
M&R LE protein marker with defined molecular weights.
The M&R LE leader peptide (M2R2, 15 kDa) was assembled with D2, MBP or Nus to generate 25–120 kDa molecular weight marker proteins (M&R LE protein marker). (A) The expression of individual (left) or mixed (right) M&R marker proteins was determined by staining with anti-His tag secondary antibody. The binding of secondary antibodies to individual (left) or mixed (right) M&R marker proteins was determined by staining with HRP-conjugated (B) anti-mouse or (C) anti-rabbit IgG Fc secondary antibodies. The relative molecular weights (kDa) of a commercial prestained marker are indicated on the left and right. The chemiluminescence was captured by an UVP BioImaging system.
Table 2.
Commercial secondary antibodies tested for recognition of the M&R LE protein marker in Western Blots.
Fig 5.
Relationship between molecular weight and electrophoretic mobility of M&R LE protein marker.
The relative mobilities of the M&R LE protein marker (○) or commercial prestained markers (●) were plotted against the logarithmic molecular weights of each marker protein. Simple linear regression was used to determine the best fit lines. The R2 value were calculated by SigmaPlot 10 software.