Goat anti rabbit igg antibody

Samples were boiled, and individual proteins were resolved by SDS-PAGE. After transfer, the PVDF membranes were decorated with the following primary antibodies: anti-HA antibody (BioLegend 901501, mouse), anti-TAP antibody (Invitrogen CAB1001, rabbit), anti-tubulin antibody (Abcam ab184970, rabbit), and anti-Sfh4 antibody (Bankaitis laboratory, Texas A&M University, rabbit). The blots were subsequently developed using the following HRP-conjugated secondary antibodies: goat anti-mouse IgG antibody (Millipore AP124P) and goat anti-rabbit IgG antibody (Millipore AP307P). Immunoblot images were acquired using Bio-Rad ChemiDoc XRS+ system. Relative band intensities on the blots were quantified using ImageJ (version 1.51s, National Institutes of Health).

The P₄ concentrations in the plasma were determined using the enzyme immuno-assay (EIA) methods described previously [19 (link)]. The
E₂ levels in plasma were also measured using the EIA methods as described by Isobe [18 (link)], with some modifications. Briefly, 75
µl of extracted estradiol-17β standard or the sample and 25 µl of the 1:10,000 diluted estradiol-17β antibody (ab215528, Abcam, Cambridge, UK) were
applied to the wells of plates that had been previously coated with goat anti-rabbit IgG antibody (Millipore; Merck & Co., Inc., Kenilworth, NJ, USA). After 1 hr incubation, 25
µl of 1:2,500 diluted estradiol-6-CMO-HRP antigen (East Coast Bio, Inc., North Berwick, ME, USA) was added to the wells. The plate was incubated for another hour and then
washed six times. The substrate solution was applied to the wells, and then, they were incubated for 30 min at room temperature. The reaction was stopped by the addition of 1 M
H₃PO₄. The optical density was calculated at 450 nm. Sensitivity was 1.6 pg/ml for E₂ and 0.5
ng/ml for P₄. The intra-assay and inter-assay CVs were 11.4% and 6.7% for E₂ and 8.6% and 3.2% for P₄, respectively.