Category Archives: Folate

Purification and Characterization of Intrauterine Folate: DISCUSSION(4)

It was surprising that FBP could not be detected in allantoic fluid by immunoblotting. Our best explanation is that the concentration of FBP in allantoic fluid is below the detection limit of our immunoblotting method. Subsequent measurements using RIA (unpublished … Continue reading

Purification and Characterization of Intrauterine Folate: DISCUSSION(3)

Both immunoblot analysis and [3H]folic acid-binding studies indicated that the amount of FBP increased dramatically between Days 10 and 15 of pregnancy. This period of pregnancy is concurrent with increased secretion of other proteins by the uterus, including uteroferrin and … Continue reading

Purification and Characterization of Intrauterine Folate: DISCUSSION(2)

Human folate receptor gamma lacks the glycophosphatidylinositol linkage site and is thought to be a secreted protein, possibly involved in folate transport. Folate receptors can be released from cells by proteolytic cleavage of the protein N-terminal to the glycosylphosphatidylinositol anchor … Continue reading

Purification and Characterization of Intrauterine Folate: DISCUSSION(1)

This study is the first to describe a folic acid-binding protein in the intrauterine environment in pigs. Results indicate that this protein is similar to other known FBPs from other species, that it has high affinity for folic acid, and … Continue reading

Purification and Characterization of Intrauterine Folate: RESULTS(2)

Assuming amo-lecular mass for FBP of 30 000 Da and a single binding site, only 21% of the FBP actually bound [3H]folic acid. The loss of binding may be the result of the repeated low-pH treatments during purification and processing … Continue reading

Purification and Characterization of Intrauterine Folate: RESULTS(1)

Typical chromatograms resulting from purification of FBP from pseudopregnant flushings and allantoic fluid are illustrated in Figure 1. Yield of FBP from pseudopregnant flushings was low (< 1 ^g), necessitating the use of silver staining to visualize the resultant protein … Continue reading

Purification and Characterization of Intrauterine Folate: MATERIALS AND METHODS(5)

At slaughter the uterus was recovered, and each uterine horn was flushed with 20 ml Minimum Essential Medium containing one tenth the normal amount of leucine. Pregnancy was confirmed by the presence of concep-tuses. Flushings from the two uterine horns … Continue reading

Purification and Characterization of Intrauterine Folate: MATERIALS AND METHODS(4)

Antibody specificity to FBP was subsequently tested by immunoblotting. Purified allantoic fluid FBP (10 ^g), allantoic fluid proteins, fetal liver membrane proteins, endometrial membrane proteins, and uterine flush proteins were all subjected to SDS-PAGE; the gels were then blotted onto … Continue reading

Purification and Characterization of Intrauterine Folate: MATERIALS AND METHODS(3)

Scatchard Analysis FBP purified from allantoic fluid was used for Scatchard analysis of folate binding. The FBP was stripped of folate by dialysis against 50 mM glycine, pH 2.8, and then further dialyzed against 50 mM Tris, pH 8.2. Concentrations … Continue reading

Purification and Characterization of Intrauterine Folate: MATERIALS AND METHODS(2)

Initially, 10 ^Ci [3H]folic acid (specific activity 50 Ci/mmol; Amersham, Arlington Heights, IL) was added before G-100 chromatography to determine those fractions that contained FBP, and an aliquot from each fraction was then subjected to scintillation counting. In subsequent runs, … Continue reading