Article Title: Normal Development and Fertility of Knockout Mice Lacking the Tumor Suppressor Gene LRP1b Suggest Functional Compensation by LRP1
Figure Lengend Snippet: Affinity purification of brain lysate on LRP1b ectodomains bound to anti-FLAG agarose. FLAG-tagged ectodomains containing the ligand-binding region II, III, and IV of LRP1b were purified from the supernatant of stably transfected 293S cells using anti-FLAG agarose. The immobilized ectodomains and FLAG-tagged BAP as a control were incubated with the same amount of brain lysate overnight and then washed extensively. Bound proteins were eluted with 3xFLAG peptide and separated by SDS-PAGE (4 to 15% acrylamide). The nonbinding fraction (flowthrough) was diluted 1:200 and loaded as a control. Protein bands were visualized with Coomassie (ectodomains II and IV) or silver staining (ectodomain III). Bands were cut out and analyzed by reversed-phase nano-HPLC-ion-trap mass spectroscopy. Proteins identified in each band by mass spectroscopy are indicated. Ecto, ectodomain; LamRP, laminin receptor precursor.
Article Snippet: As a control, bacterial alkaline phosphatase (BAP) containing three N-terminal FLAG epitopes (Sigma) was bound to M2 anti-FLAG agarose and incubated the same way with brain tissue.
Techniques: Affinity Purification, Ligand Binding Assay, Purification, Stable Transfection, Transfection, Incubation, SDS Page, Silver Staining, Mass Spectrometry