Goat anti rabbit igg hrp

Unless otherwise noted, cells of the late exponential phase were harvested, washed with phosphate buffered saline (PBS), resuspended in the same buffer, and sonicated. Protein concentrations of the cell lysates was determined by the bicinchoninic acid assay (Pierce Chemical). For heme-staining, the cell lysates were separated on SDS-PAGE using 12% polyacrylamide gels and stained with 3,3′,5,5′-tetramethylbenzidine (TMBZ) as described elsewhere43 (link). Immunoblotting analysis was performed essentially the same as previously described44 (link). Proteins separated on SDS-PAGE were electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane according to the manufacturer’s instructions (Bio-Rad). The gels were blotted for 2 h at 60 V using a Criterion blotter (Bio-Rad). The blotting membrane was probed with rabbit polyclonal antibodies against NrfA. The goat anti-rabbit IgG-HRP (horseradish peroxidase) (Roche Diagnostics) was used as the secondary antibody (1:5,000) and the signal was detected using a chemiluminescence Western blotting kit (Roche Diagnostics) in accordance with the manufacturer’s instructions. Images were visualized with a UVP imaging system.

Digoxigenin-UTP-labeled riboprobes were synthesized according to the manufacturer’s instructions (Roche), and in situ hybridizations were performed as described previously [45 (link)]. The color reaction was conducted using the NBT/BCIP substrate (Roche). To minimize the variation between the control and experimental groups, we used embryos produced by a single pair of parents and always used the same number of embryos for the control and experimental groups. The groups were compared under precisely same experimental conditions at the same time, and color reactions were initiated and stopped at precisely the same time. For immunohistochemistry, zebrafish embryos were blocked in 5 % goat serum and incubated with a rabbit phospho-histone H3 antibody or rabbit monoclonal antiactive caspase-3 (1:200, Abcam). Goat antimouse IgG HRP or goat antirabbit IgG HRP (Roche) was used for detecting the primary antibodies, and DAB was used as a substrate for the secondary antibody-conjugated HRP (Amresco). The embryos were mounted in Vectashield mounting medium (Vector Laboratories, Inc.).

Unless otherwise noted, mid-log phase cells were harvested, washed with phosphate buffered saline (PBS), resuspended in the same buffer, and sonicated. Protein concentrations of the cell lysates were determined by the bicinchoninic acid assay (Pierce Chemical). The cell lysates were resolved by SDS-PAGE using 12% polyacrylamide gels and stained with 3,3′,5,5′-tetramethylbenzidine (TMBZ) as described elsewhere (Thomas et al., 1976 (link)).
Immunoblotting analysis was performed essentially as previously described (Dong et al., 2012 (link)). Proteins separated by SDS-PAGE were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes according to the manufacturer’s instructions (Bio-Rad). The gels were blotted for 2 h at 60 V using a Criterion blotter (Bio-Rad). The blotting membrane was probed with a rabbit polyclonal antibody against S. oneidensis Crp (Dong et al., 2012 (link)). Goat anti-rabbit IgG-HRP (horseradish peroxidase; Roche Diagnostics) was used as the secondary antibody (1:5,000) and the signal was detected using a chemiluminescence Western blotting kit (Roche Diagnostics) in accordance with the manufacturer’s instructions. Images were visualized with a UVP imaging system.