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Caroline Pasquay, Annabella Janise, Birgit Lorenz, Markus Preising; Characterisation of a novel antibody against the loop between TMD 3 and 4 of human Bestrophin1. Invest. Ophthalmol. Vis. Sci. 2013;54(15):702.
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Purpose: Bestrophin1 is part of an integral membrane protein complex located in the basolateral membrane of the retinal pigment epithelium. The gene is mutated in Best vitelliform macular dystrophy (VMD), an autosomal dominant macular degeneration with highly variable expressivity and reduced penetrance. We want to investigate whether mutant bestrophin1 subunits form complexes with wildtype protein subunits to get further information about the function of mutant protein complexes which is not well understood up to now. For this purpose we constructed a novel antibody against the loop between transmembrane domains (TMD) three and four and confirmed its applicability in Western blotting.
Methods: An alignment with the four isoforms of human bestrophin (1-4) was performed. A human bestrophin1 specific sequence stretch (AA204-220: PILLQSLLNEMNTLRTQ) within the loop between TMD 3 and 4 was chosen as antigen and an oligopeptide was synthesized. The antibody was raised against the oligopeptide in chicken and affinity purified by a commercial supplier. The antibody specificity was confirmed by an ELISA against the oligopeptide MDCK II cells were seeded on transwell filters and cultured in high glucose DMEM supplemented with 10% FCS and 5 times penicillin/streptomycin at 37°C and 5% CO2. His-tagged BEST1 cloned into a pcDNA3(-) vector was transfected into MDCKII cells using RotiFect® according to the manufacturers’ instructions at subconfluency. Cells were harvested 24 h after transfection and lysed. After lysis proteins were extracted and protein preparation took place after a modified protocol according to Bordier (JBC 1981). SDS-PAGE and Western blotting were performed following a standard protocol.
Results: The antibody showed a single specific band corresponding to a molecular weight of approx. 67 kD on SDS-PAGE and Western blotting. This corresponds well with the monomer size of human bestrophin1. Counterstaining by a commercially available anti-His antibody labelled the identical band.
Conclusion: A novel antibody (cab-BEST1) was raised in chicken against the intracellular loop between TMD 3 and 4 of human bestrophin1. The antibody was specific for human bestrophin1 in Western blots and will be useful in testing the function of the N-terminal half of bestrophin1. Further data on its applicability in immunohistochemistry will be available at the meeting.
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