Rabbits were immunized with a synthetic peptide representing amino acid residues 95 to 108 (GKPIPNPLLGLDST) of RNA polymerase alpha subunit of simion virus 5 conjugated to KLH. Antibody was isolated by affinity chromatography using the peptide immobilized on solid support and conjugated to horseradish peroxidase (HRP). Antibody concentration was determined by extinction coefficient prior to conjugation: absorbance at 280 nm of 1.4 equals 1.0 mg of IgG. Molar enzyme/antibody protein ratio is 4:1.
2 - 8°C
Detection of V5-tagged Protein by western blot. Samples: 200, 100, or 50 ng of E. coli whole cell lysate expressing a multi-tag fusion protein. Antibodies: Affinity-purified, HRP-conjugated, rabbit anti-V5 antibody A190-120P used for WB at 0.2 µg/ml (1:5,000). Detection: Chemiluminescence with an exposure time of 30 seconds.