Introduction 1999 1984 1983 1997 1998 1999 2006 2004 2005 2000 1998 2004 2006 2006 2002 2006 1 1997 2002 1996 1993 2004 1997 Materials and methods Materials 1 2a 2a Bam Xho Eco Hin Molecular cloning and transfection 1 Bam Xho Hin Xho 1 2006 Western blot Cells were washed and scraped in PBS, spun down and lysed in RIPA buffer with protease inhibitor. Protein concentration was determined using the BCA kit according to the manufacturer’s protocol. Ten micrograms of protein were loaded onto a 4–12% NuPAGE® Bis–Tris Gel. Electrophoresis was carried out at 200 V for 40 min in NuPAGE® MES running buffer. Protein was transferred to Invitrolon™ PVDF blotting membranes at 30 V for 1 h. The membrane was blocked for 1 h at room temperature in 50 mg/ml non-fat dry milk in PBS followed by overnight incubation at 4°C with a dilution of 1:5,000 for mouse anti-HisG. After washing with PBS-Tween (0.1%), the membrane was incubated with a 1:20,000 dilution of goat anti-mouse-HRP IgG at room temperature. Detection was done using BM Chemiluminescence Blotting Substrate and Hyperfilm ECL. Immunocytochemistry Cells (40,000 cells/well) were plated on day 1 in 96 wells black, clear bottom plates or eight well chamber slides. The next day, the medium was changed to serum-free medium. After incubation overnight, cells were stimulated with the indicated ligand in serum-free medium for 30 min at 37°C, unless otherwise indicated. The stimulation solutions were removed, and cells were fixed by applying 4% v/v formaldehyde in PBS for 15 min at room temperature. After washing three times with PBS, cells were incubated with the first antibody for 1 h at room temperature. Cells were washed again three times with PBS and, if necessary, incubated with the second antibody for 1 h at room temperature. Cells were washed three times with PBS followed by measurements on a Victor2 (Wallac, Perkin Elmer). The following antibody combinations were used: mouse anti-HisG 1:200 with AlexaFluor® 488 goat anti-mouse 1:500, mouse anti-HisG-HRP 1:200 and mouse anti-HisG 1:200 with goat anti-mouse-HRP 1:500. In case an HRP-bound antibody was used, either ABTS solution (50 μg/ml) or BM Chemiluminescence Blotting Substrate was added to generate an absorbance or luminescence signal, respectively. For the absorbance signal, a 405-nm filter was used; for the fluorescent signal, a 490-nm excitation and a 535-nm emission filter were used. Data analysis t P Results 1 1 1 1 2 1 1 2 1 50 1 1 n 2006 Fig. 1 1 lane 1 1 lane 2 1 arrow  Fig. 2 1 a 1 b  1 1 2004 3 3 1 4 1 1 5 Fig. 3 1 left panel right panel  Fig. 4 1  Fig. 5 1 first bar 1 second bar 1 n  Choice of detection method 6 6 6 6 6 1 50 n 50 n 7 Fig. 6 1 n a b c d e asterisk pound sign  Fig. 7 closed squares open squares 1  Discussion 1997 2002 1996 1993 1 2004 1997 2002 1996 1993 1 While our technique does not allow following internalisation in real time, the ability to process a large number of samples compensates for that by studying multiple time points. The only limitation to our method is the inability to measure internalisation of endogenous receptors. However, this would be possible when specific receptor antibodies are available, which is not yet the case for our model receptor. In conclusion, we have validated a method to quantitatively measure receptor internalisation in which we use an N-terminal HisG-tag combined with immunocytochemistry. It proves to be sensitive enough to discriminate between different receptor ligands in a fast and non-radioactive way.