Capturem Protein G Miniprep columns were used according to the Capturem Protein G Miniprep protocol. First, the columns were equilibrated with 300 µl of Pierce Protein G Binding Buffer and then centrifuged at 1,000g for 1 min. Serum samples from sheep, goat, rat, mouse, human, rabbit, and horse (250 µl each) were diluted in 1 ml of Protein G binding buffer, and 600 µl of sample was loaded on an equilibrated column, followed by centrifugation at 1,000g for 1 min. The loading process was then repeated with another 600 µl of sample. The columns were then washed with 800 µl of Protein G binding buffer at 1,000g for 1 min. The bound antibody was then eluted with 300 µl of elution buffer (0.1 M glycine, pH 2.5) into a tube containing 30 µl of neutralization buffer (1 M Tris, pH 8.5) to neutralize the eluted antibody.
Capturem Protein G Maxiprep columns were used according to the Capturem Protein G Maxiprep protocol. First, the columns were equilibrated by loading 6 ml of Pierce Protein G binding buffer and centrifuging at 2,000g for 3 min. Next, 6.3-ml samples of mouse hybridoma supernatant from a bioreactor were diluted to 25 ml in Protein G binding buffer and then loaded onto the equilibrated columns by centrifugation at 2,000g for 3 min. We collected samples of the flowthrough for SDS-PAGE (Lane FT1 in Figure 1), then centrifuged the 25-ml samples through the columns a second time and collected another set of samples for SDS-PAGE (Lane FT2 in Figure 1). The columns were then washed once with 20 ml of Protein G binding buffer at 2,000g for 3 min. Lastly, four consecutive elutions were performed, each with 1 ml of elution buffer. The eluted fractions were analyzed using SDS-PAGE and quantified by measuring the absorbance at 280 nm with a NanoDrop 2000 spectrophotometer.
For the comparison of the Capturem 96-well plate and the Protein G resin 96-well plate, hybridoma supernatants from each clone were loaded onto each plate following the protocol for each product. The plates were first equilibrated, and then the supernatants were loaded and washed according to the protocols. Finally, the monoclonal antibody clones were eluted with 600 µl of elution buffer plus 60 µl of neutralization buffer in a collection tube.
To test the reproducibility of our 96-well plates, we loaded eight identical samples of two different hybridoma supernatants and animal sera into individual wells. Again, we equilibrated, loaded, washed, and eluted according to the protocol using Pierce Protein G Binding Buffer for all binding and washing steps. Eluates from various samples were quantified by measuring the absorbance at 280 nm with a NanoDrop 2000 spectrophotometer.