Alexa fluor 680 goat anti mouse igg secondary antibody

Bacterial cell lysates, glycoproteins and non-glycosylated AcrA were resolved on 10% SDS-PAGE. After transfer to a nitrocellulose membrane (Hybond-ECL, GE Healthcare), analysis by immunoblotting was performed using 1:200 O157 1E10, 1:1000 O157 3F10, 1:150 O157 10G2, 1:100 O145 2H6, 1:1000 O145 4C8 or 1:100 O145 4E6 hybridoma supernatant concentrates. Bound mAbs were recognized with Alexa Fluor 680 goat anti-mouse IgG secondary antibody (Invitrogen) at a 1:20000 dilution and visualized with an Oddysey Infrared Imager (Li-Cor).

Food protein extracts, purified β-casein (Sigma-Aldrich) and fresh milk -UHT skim milk (milk), raw milk (a kind gift from Gabriela Rodriguez, INTI lacteos, Buenos Aires, Argentina), 10% skim milk powder (milk powder) and goat milk-, were resolved on 10% SDS-PAGE. After transfer to a nitrocellulose membrane (Hybond-ECL, GE Healthcare), analysis by immunoblotting was performed using 0.02 μg/ml 1H3 or 0.2 μg/ml 6A12 purified monoclonal antibodies in blocking buffer (5% BSA in TBS). Bound antibodies were recognized with an HRP goat anti-mouse IgG secondary antibody (Sigma-Aldrich) at a 1:6000 dilution in blocking buffer, or with Alexa Fluor 680 goat anti-mouse IgG secondary antibody (Invitrogen) at a 1:20000 dilution in blocking buffer. The signal was visualized with enhanced chemiluminiscence reagent (GE Healthcare) and CL-XPosure Films (Thermo Scientific), or with an Oddysey Infrared Imager (Li-Cor). Densitometric analysis was performed using the Li-Cor Image Studio Lite software.