How does R & D Antibodies perform western blots using mouse monoclonal antibodies?
1. After SDS-PAGE (on either 4-15% gradient gels or single percentage gels, such as 7.5% or 10% gels) and electrophoretic transfer to PVDF membrane, the membrane is blocked overnight with 4% normal goat serum using TBS/Tween-20 buffer as diluent. 2. Wash x 2 for 5 min with TBS/Tween-20. 3. Apply the mouse monoclonal antibody after dilution to an appropriate volume (at least 1:20 for culture supernatant or 1:500 for ascites – see Note below). Use 2% normal goat serum in TBS/Tween-20 as buffer for the primary antibody. Let the primary antibody bind for 2-4 hours. 4. For blocked antibody controls, add 150 nmoles of the peptide immunogen or 150 μgm of the protein antigen to 6.0 ml of the diluted monoclonal antibody. Preincubate for 60 min. before applying to the membrane. This should be sufficient for 3 blocked control lanes. DO NOT ADD THE PEPTIDE OR PROTEIN TO THE STOCK MONOCLONAL ANTIBODY. 5. Wash x 3 for 5 min with TBS/Tween-20. 6. Apply affinity purified HRP-goat anti-mouse IgG or IgM
