Immunoprecipitation was performed on purified TAMRA-BSA. The antigen:antibody complex was formed by binding 5µg protein with 10µg of mouse monoclonal antibody recognizing TAMRA (Product # MA1-041) or Mouse IgG, whole molecule ( Product # 31202) overnight on a rocking platform at 4°C. The immune-complex was then captured on 50µl Protein A/G Plus Agarose (Product #20423) . Captured immune-complexes were then washed extensively and proteins eluted with 5X Reducing Sample Loading Dye (Product # 39000). Samples were then resolved on a 4-20% Tris-HCl polyacrylamide gel. Proteins were imaged using Typhoon 9410 Imager at 532nM , Normal Sensitivity.
Western blot analysis was performed by loading various amounts of purified TAMRA-BSA protein onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% Milk/TBST for at least 1 hour. Membranes were then probed with a mouse monoclonal antibody recognizing TAMRA (Product #MA1-041) at a dilution of 1:1000 for 1hour at room temperature on a rocking platform. Membranes were then washed in TBS-0.1%Tween 20 and probed with a goat anti-mouse-HRP secondary antibody (Product #31430) at a dilution of 1:20,000 for at least one hour. Membranes were washed and chemiluminescent detection was performed using Pierce Super Signal West Pico (Product #34087).