Rabbits were immunized with 6-His (HHHHHH) conjugated to KLH. Antibody was isolated by affinity chromatography using the peptide immobilized on solid support and conjugated to horseradish peroxidase (HRP). This antibody does not detect all N-terminal 6-His tagged proteins. 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
Phosphate Buffered Saline (PBS) containing 0.2% BSA and 0.05% Pro-Clean 400
Detection of 6-His-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-6-His antibody A190-114P used for WB at 0.04 µg/ml (1:25,000). Detection: Chemiluminescence with an exposure time of 3 seconds.