Dear Muhammad,

The required assay is described in the following text:

Folate microbiological assay of fetal liver and placenta

Folate content was measured with Lactobacillus rhamnosus (ATCC 7469) in a 96-well microplate using randomly selected fetal liver (n = 10) and placental tissues (n = 8) [18]. After washing with phosphate-buffered saline (pH 7.0), the whole placenta was homogenized with 0.1 M potassium phosphate buffer containing ascorbic acid (58.8 mmol/l) and centrifuged (240 × g for 10 min, 4℃). The supernatant was incubated with rat serum folate conjugase (8 h, 37℃, pH 7.0) to hydrolyze polyglutamyl folates to monoglutamyl folates, which are readily utilized by the bacteria. After heating (10 min, 100℃) and subsequent centrifugation (2,450 × g, 10 min), the supernatant was directly used for folate assay.

Western blotting analysis in the placenta

Randomly selected whole placental tissues (n = 10) were homogenized in RIPA Cell Lysis Buffer (pH 7.5, GenDepot, Barker, TX, USA) with ProteoBlock Protease Inhibitor Cocktail (Fermentas, Burlington, Canada) and centrifuged (16,600 × g for 15 min, 4℃). The protein content of the supernatant was determined using a bicinchoninic acid (BCA) Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). Then, 15 µg of protein was applied onto 10% SDS-polyacrylamide gel and electro-transferred onto a nitrocellulose transfer membrane (Whatman, Dassel, Germany), which was probed with primary antibodies against either FRα or β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA); horseradish-peroxidase-linked antibody was used as a secondary antibody (Santa Cruz Biotechnology). The blotted membranes were visualized with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific), and the density of each band was quantified using LAS-3000 and MultiGauge V3.0 densitometer (Fujifilm, Tokyo, Japan).

Statistical analysis

Data were analyzed using SPSS V12.0 (Chicago, IL, USA) and are expressed as mean ± SEM. The differences of among the two groups of data were evaluated by Student t-test. A P value < 0.05 was accepted as significant.

To view the full publication, please use the following link:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3085799/

Hoping this will be helpful,

Rafik

thank you Mr. Rafik Karaman