Goat anti human igg 680rd antibody

For protein analysis, human antibodies were purified from supernatants of transfected cells with a Protein A antibody purification kit (Montage Antibody Purification Kit with PROSEP-A media, Millipore). Purified antibodies were separated in precast Bis-Tris gels (Invitrogen) under either reducing or nonreducing conditions. Proteins were transferred to Immobilon-FL PVDF transfer membranes (Millipore). Membranes were blocked for 1 hr in Odyssey Blocking Buffer (Li-Cor Biosciences), incubated with a goat anti-human IgG 680RD antibody (Li-Cor Biosciences), and washed. Protein bands were visualized on the Li-Cor Odyssey CLx.

To construct the PSMA-DMAb , the genes of both the variable heavy (V_H) and variable light (V_L) fragments of a human anti-PSMA mAb were examined, optimized, and constructed through the use of synthetic oligonucleotides with several modifications to improve expression as previously described [15 (link)]. DNA was formulated in water for subsequent administration into mice. An empty pVax1 expression vector was used as a negative control. Cells (293T) were transfected with the PSMA-DMAb plasmid and confirmation of PSMA-DMAb binding to recombinant human PSMA was carried out by Western blot analysis. Briefly, recombinant PSMA protein (R&D systems) was run on an SDS-PAGE gel and transferred to Immobilon-PVDF membrane (EMD Millipore). Membranes were blocked for 1 h in blocking buffer (Li-Cor Biosciences) and then incubated for 1 h with either commercial anti-PSMA mAb (R&D systems), pooled day 14 sera from PSMA-DMAb plasmid-injected mice, or supernatants from PSMA-DMAb plasmid-transfected 293T cells. Membranes were washed and then incubated for 1 h with a goat anti-human IgG 680RD antibody (Li-Cor Biosciences) and washed. Protein bands were visualized by scanning membranes with a Li-Cor Odyssey CLx scanner [19 (link)].