Monoclonal Antibodies Specific for Disease-Associated Point-Mutants: Lamin A/C R453W and R482W
Figure 4
R482W antibody recognizes a discontinuous epitope in the lamin A/C Ig-fold.
(A) Equal amounts of peptide pR482 (472QNGDDPLLTYRFPPKFTLKAG492), pW482 (472QNGDDPLLTYWFPPKFTLKAG492), pR453 (449GKFVRLRNK457), pW453 (449GKFVWLRNK457) and equal amounts of recombinant HBcAg Ig-fold wt, HBcAg Ig-fold R453W, and HBcAg Ig-fold R482W were spotted onto PVDF membranes. Blots were incubated either with anti R482W antibody (upper left panels) or with anti R453W antibody (upper right panels). Anti lamin A/C (middle panels) and Ponceau S staining (bottom panels) confirmed equal spotting of recombinant HBcAg Ig-fold proteins or peptides, respectively. (B) 12.5% SDS-PAGE of bacterial lysates expressing His-tagged HBcAg Ig-fold R482W fragments of increasing size followed by Western blot analysis with R482W antibody (left panel). Equal amounts of recombinant proteins were confirmed by incubation with anti-His antibody (right panel). Numbers correspond to amino acids of lamin A/C.