Binding of omeprazole to protein targets identified by monoclonal antibodies
Fig 5
Time and temperature dependent omeprazole-protein complex formation.
(A) Incubation of 1mM omeprazole with non-cysteine containing protein, casein, at 65˚C indicates a time dependent (0.5, 1, 5, 10, 15, 30, 45, 60, 75, 90, and 120 minute points) increase in omeprazole-casein complex which was recognized by the antibody. (B) Omeprazole (1mM) reaction with purified proteins, casein, TSP1, KLH, for 2hr at 4, 22, 37, and 65˚C show that there is increased complex formation with increased temperature. Note that the lowest molecular weight TSP1 band reacted with the antibody without omeprazole, representing a non-specific reaction; higher molecular weight bands are specific for omeprazole treatment. There is higher molecular weight oligomer formation with both the time and temperature dependent studies; baseline state (●) and omeprazole-dependent higher molecular weight states (׀) are marked. Oligomerization was especially pronounced in TSP1. There was no reducing agent in sample loading buffer, and samples were not boiled. Protein molecular masses are as follows: for casein (monomer at 27kD), TSP1 (baseline multimer >250kD), and KLH (monomer at >250kD). 5C3 antibody (1:50 dilution) was used for the detection of the samples.