Alexa fluor 680 donkey anti rabbit igg secondary antibody

Supernatant proteins from exponential phase THY broth GAS cultures were concentrated by ethanol precipitation and resuspended in SDS-PAGE buffer at 1/20th the original volume. Protein samples were separated on 12% Tris-HCl gels before transferring to membrane and using in Western blot analysis with a custom rabbit anti-streptokinase (SKA) polyclonal antibody (made by Pacific Immunology Inc), a commercial rabbit anti-SLO/SPN polyclonal antibody (American Research Products Inc), a custom rabbit anti-GRAB polyclonal antibody (made by Pacific Immunology Inc), and a custom rabbit anti-Spd polyclonal antibody (made by Pacific Immunology Inc), as primary antibodies. After washing, Alexa Fluor 680 donkey anti-rabbit IgG secondary antibody (Molecular Probes) was used (1:10,000 dilution), and the fluorescent signal was detected using a Li-Cor Odyssey Near-Infrared System.

Protein samples were separated on a standard 10% SDS-PAGE gel with the addition of 100 μM Phos-tag^™ Acrylamide AAL-107 (Wako Pure Chemicals) and 100 μM MgCl₂ (Sigma-Aldrich). Equal amounts of cytoplasmic GAS protein samples (70 μg) were loaded onto the SDS-PAGE gel and electrophoresis set at 100 volts for >2 hr at 4 °C. Samples were then transferred to PVDF membrane via wet transfer at 400 constant mA. Analysis of proteins was accomplished with use of a custom rabbit anti-CovR antibody as previously described (Horstmann et al., 2015 (link)). After washing, Alexa Fluor 680 donkey anti-rabbit IgG secondary antibody (Molecular Probes) was used (1:10,000 dilution), and the fluorescent signal was detected using a Li-Cor Odyssey Near-Infrared System.

Protein samples were separated on a 6% SDS-PAGE gel. For use as a loading control the membrane was stained using the MemCode Reversible Protein Stain Kit (Pierce). Subsequently, the membrane was used in Western blot analysis using monoclonal T2 sera (TransEurope Chemicals; used at a 1:500 dilution) as the primary antibody. Monoclonal T2 sera reacts against the major pilus protein of serotype M2 GAS strains. After washing, Alexa Fluor 680 donkey anti-rabbit IgG secondary antibody (Molecular Probes) was used (1:10,000 dilution), and the fluorescent signal was detected using a Li-Cor Odyssey system. For densitometric analysis of the Western data we utilized Li-cor Image Studio Lite Software.