Anti c myc cy3 antibody

Peptide SPOT arrays were synthesized using F-moc chemistry on nitrocellulose membranes at the MIT Biopolymers facility using an Intavis AutoSpot robot. Peptide spots were cut from the membrane, hydrated in 100% methanol, transferred to TBS (50 mM Tris, pH 8.1, 100 mM NaCl, 0.01% Triton X-100) with 1% bovine serum albumin (BSA), here called blocking buffer, and incubated at room temperature for ∼10 minutes. Membranes were then incubated with 10 ml of 1 µM or 100 nM c-Myc-tagged receptor (sequences in Table S6) in TBS for 1 h at room temperature. Membranes were then rinsed 3× with blocking buffer and then incubated with anti-c-myc-Cy3 antibody (Sigma Aldrich C6594) diluted 100-fold in blocking buffer for 1 hour at room temperature. Membranes were rinsed 3× with blocking buffer and scanned on a Typhoon 9400 (GE Healthcare). Images were analyzed with ImageQuant (GE Healthcare), and Cy3 intensity at 580 nm was averaged over a circular area that was equal in size for all spots for a given membrane. Typically, 5–10 known binders and their negative controls were included.

BON cells (subclone #7) were kindly provided by Dr Courtney M. Townsend (Department of Surgery UTMB, Galveston, TX 77551, USA) [16] (link), [17] (link). They were grown in DMEM/F-12 (Ham) without phenol red (GIBCO, Invitrogen Corporation) supplemented with 10% fetal bovine serum (Hyclone, Perbio) and antibiotics (100 U penicillin/mL and 100 µg streptomycin/mL, Invitrogen), at 37°C in a humidified incubator with 5% CO₂. Cells were transiently transfected with pCMV-TagOR1G1 or pCMV-TagOR17-40 using jetPEI™(Polyplus-transfection) according to the manufacturer’s instructions. OR expression at the cell surface was checked by immunofluorescence microscopy using the monoclonal anti-cmyc-Cy3 antibody (C6594, Sigma-Aldrich) on non-permeabilised cells.
LNCaP cells were purchased from ATCC (Clone FGC, No. CRL-1740™) at passage 19, and grown in RPMI 1640 medium (ATCC, No. 30-2001) supplemented with 10% fetal bovine serum (ATCC, No. 30-2021), at 37°C in a humidified incubator with 5% CO₂.