ASAS/ADSA Growth and Development

To determine the elution pattern and electrophoretic characterization of granular-secretory fraction of the bovine fetal cotyledons, an experimental procedure was developed. A multi-proteic complex extract of the granular-secretory fraction of the bovine fetal cotyledons, was frozen at -20◦C, then was centrifuged at 46,000 x g for 60 min at 4 ◦C. After that, the supernatant was removed and saved by further use. The pellet was washed with a 0.05 M NH4HCO3 buffer solution at pH 7.8 and then was centrifuged at 46,000 x g at 4◦C for 60 min, the supernatant also was removed. Both supernatants were mixed and suspended together, then recovered protein was quantified reading in spectrophotometer at 595 nm. The suspension was applied to a 5 x 70 cm column of Sphadex G-75. The protein was eluted with a pH 7.8 buffer solution of 0.05M NH4HCO3 at 4◦C. From the elution peak observed in the elution pattern, aliquots were obtained for electrophoretic characterization. The fractions with MW, were divided in aliquots of 50 mL or less. 0.125g of sacarose were added for each aliquot. Then were frozen at -20 ◦C and lyophilized at -44 x 10−3 mBar. To characterize the electrophoretic pattern, SDS-PAGE with 12.5% gels were used. Protein recuperation of elution the process was 82.9%. The elution pattern showed three different peaks; the first one named peak A, has the 32.7% of the protein in 150 mL of eluyent; in a second peak (B) 62% of protein was obtained in 165 mL of buffer; in a third peak (C), 4.8% of protein was obtained in 140 mL of eluyent. The strip pattern of electrophoresis, produced three main regions, the first one grouped three proteins with the higher MW ranks from 42 to 95 kDa, and a second one constituted with proteins of intermediate MW (33.5 kDa); and third one grouping three proteins complex with the lower MW ranking from 20.5 to 28.5 kDa. It is concluded that the procedure described is able to produce partially purified placental bovine proteins usable in bioassays and ultrapurification process.