Goat anti rabbit igg ap conjugate

Total HeLa, crATIC, crGART, and crGART/K1-GART (transfected cells) protein (30 μg/lane) was separated by SDS-PAGE (8–16% gradient tris-glycine gel) for 40 minutes at ~225 V. Gels were wet-blotted to nitrocellulose (Amersham Protran 0.45 μM NC, buffer: 25 mM Tris, 200 mM glycine, 20% methanol) at 200 mA for 1 h. Blots were blocked 30 minutes in block solution (1% BSA, 0.1% Tween-20, 1X TBS, 0.01% sodium azide, pH 7.4) then incubated for 1.5 h at RT with either CD36 (Abcam ab133625, 1:500 dilution) or PPARγ (Cell Signaling 2430S, 1:250 dilution) and β-actin (Cell Signaling 8457, 1:1000 dilution) rabbit primary antibodies in primary Ab solution (5% BSA, 0.1% Tween-20, 0.01% sodium azide, 1X TBS, pH 7.4). Blots were washed 3X for 5 minutes in 1X TBS. Blots were then incubated in goat anti-rabbit IgG-AP conjugate (Bio-Rad, 170–6518) diluted 1:10,000 in block solution for 1 hour. Three more washes (5 minutes each in 1X TBS) were performed, then blots were treated with CDP-Star® Substrate with Nitro-Block-II™ Enhancer (Applied Biosystems, T2218) for five minutes and visualized using a Bio-Rad Chemidoc Imaging System. See S1 Raw images for unaltered immunoblot images with annotation.

Anti-FLAG M2, mouse (F1804, Sigma-Aldrich, Canada) was used to precipitate the CaSR/interactor complex. Anti-CaSR C0493, mouse (Abcam, Cambridge, MA, USA) and Anti-GAPDH mouse (Abcam, Cambridge, MA, USA) were used for western blot. Anti-VAPA (15275-1-AP, rabbit (ProteinTech, Illinois, USA) and anti-GRP78 (ab21685), rabbit (Abcam, Cambridge, MA, USA) were used for western blot and immunostaining. Goat anti-rabbit IgG-AP conjugate (1706518, BioRad) and goat anti-mouse IgG-AP conjugate (1706520, BioRad) were used as secondary antibodies for western blot. Anti-GFP antibody mouse (ab13970, Abcam) was used to immunostain GFP-CaSR. Donkey anti mouse IgG (H + L) Alexa fluor 647 (A31571, Invitrogen), goat anti-mouse IgG (H + L) Alexa Fluor 488 (A32723, Invitrogen), goat anti-mouse IgG (H + L) Alexa Fluor 555 (A-21422, Thermo Fisher Scientific) and goat anti-chicken IgY H&L Alexa Fluor 488 (ab150173, Abcam) were used as secondary antibodies for immunostaining.

Mouse monoclonal hybridoma supernatants Y3 (against the complex of K^b free heavy chain with β₂m (Hämmerling et al., 1982 (link)), 27-11-13S (against the complex of D^b free heavy chain with β₂m (Ozato and Sachs, 1981 ), hemagglutinin (HA) 12CA5 (Niman et al., 1983 (link)), and BBM.1 (Brodsky et al., 1979 (link)) were described previously. Antibodies for immunoprecipitation were rabbit anti-GFP (Abcam ab290), rabbit antisera against H-2K^b and H-2D^b (Charles River Laboratories, Kisslegg, Germany), and goat anti-rabbit IgG-AP conjugate (1706518, Biorad, Munich, Germany). Secondary antibody against the HA-antibody was donkey anti-mouse IgG Alexa Fluor 568 (a10037, Thermo Fisher Scientific, Darmstadt, Germany).