Alkaline phosphatase conjugated goat anti mouse igg

To evaluate rhAA presence in the culture medium, an indirect ELISA was performed. Microtiter plates (NUNC, Netherlands) were coated with 100 µL per well of 0.2 µg standard recombinant human activin A (Isokine™, ORF Genetics, Kópavogur, Iceland), along with rhAA produced from transgenic rice cells in a coating buffer (15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.6), and were placed at 4 °C overnight. On the following day, the wells were washed three times with PBST (PBS buffer with 0.05% Tween 20). The plate was then given 200 µL blocking buffer containing 0.1% bovine serum albumin and left at room temperature for 2 h and then washed 3 times with PBST. Subsequently, 100 µL per well of a 1:1000 dilution of inhibin β-A monoclonal antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was added. After incubating at room temperature for 2 h, the plate was washed with PBST, and 100 µL of a 1:7000 dilution of alkaline phosphatase-conjugated goat anti-mouse IgG (W4021, Promega, Madison, WI, USA) was added to wells. The microplate was incubated at room temperature for 2 h and washed three times with PBST. The color was developed by the addition of 100 µL per well of phosphatase substrates (S0942, Sigma-Aldrich, St. Louis, MO, USA). Optical density was measured at a wavelength of 405 nm using an ELISA reader (Sunrise, Tecan, Männedorf, Switzerland).

Infiltrated leaves (7 DPI) were harvested and ground in liquid nitrogen to a fine powder. For each sample, 60 mg leaf powder were extracted in 200 μl phosphate buffered saline (PBS, pH 7.4) and shaken for 15 min at room temperature. After centrifugation at 13,000 × g for 15 min at room temperature, the supernatant (PBS-soluble fraction) was transferred to a fresh tube and the pellet (insoluble fraction) was washed three times with PBS before re-extraction with buffer K (62.5 mM Tris pH 7.4, 10% glycine, 5% 2-mercaptoethanol, 2% SDS, 8 M urea). The relative quantity of total recombinant protein was estimated by directly homogenizing 60 mg leaf samples in 200 μl buffer K, sonicating for 20 s, and centrifuging as above for 10 s. Protein samples were boiled in loading buffer, separated by SDS-PAGE in 12% acrylamide gels and transferred to a nitrocellulose membrane. DsRed-fusion proteins were detected using an anti-His-tag antibody. The recombinant protein was visualized with an alkaline phosphatase-conjugated goat-anti-mouse IgG (Promega, Fitchburg, WI, USA) diluted 1:5000. Images were analyzed using Image Lab v5.1 (Bio-Rad Laboratories, Hercules, CA, USA).