Western blot analysis of hCG was performed by loading the indicated amounts of purified hCG protein extracted from human urine, and 15ul of PageRuler Prestained Protein Ladder per well onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane using the G2 Fast Blotter and blocked with 5% Milk for at least 1 hour at room temperature. hCG was detected at ~22kD using a anti hCG antibody at a concentration of 4 µg/mL in blocking buffer overnight at 4C on a rocking platform, followed by an HRP-conjugated goat anti-mouse IgG (Fc) secondary antibody at a dilution of 1:10,000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura.