Rabbits were immunized with E-Tag (GAPVPYPDPLEPR) 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 E-Tag-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-E-Tag antibody A190-133P used for WB at 0.04 µg/ml (1:25,000). Detection: Chemiluminescence with an exposure time of 10 seconds.