25 mm gd x

Total ten river water samples were collected from different locations of Kathmandu valley. Samples were collected from stagnant surface of river in a 100 ml sterile glass bottle. After removal of larger particulates by centrifugation at 3000 rpm for 30 minutes, the supernatant was slowly filtered through a syringe filter (Whatman 25 mm GD/X) with a pore size of 0.22 μm to a sterile 15 ml screw capped tube (Borosil).

Breast milk was collected by participating women using a manual breast pump (Camera M-11133, Quito, Ecuador) that dispensed milk into a 50 mL aseptic tube (Falcon, Quito, Ecuador); women were asked to wipe the breast with a clean warm cloth and sample 10 mL of milk from each breast. Samples were transported to the laboratory at 4 °C within 2-h of collection. Upon arrival, samples were deproteinated with 6% 5-sulfosalicylic acid dehydrated, and centrifuged at 3000 rpm for 15 min; serum supernatants were filtered with a 0.45-μm filter (Whatman 25mm GD/X) and stored in 5 mL cryogenic tubes in liquid nitrogen until amino acid determination at Ajinomoto Co. Inc., Institute of Life Science, Japan [15 (link)]. Breast milk samples were collected between 06:00–08:00 am, after approximately 10 h of fasting; colostrum—S1 was collected within the first week postpartum; transition milk—S2 between 13 to 17 days postpartum. In addition, two-mature milk samples were collected at 2 —S3 and/or 4 months postpartum—S4. Before AA measurements, samples were cleared with an Amicon Ultra centrifugal filter (Millipore, Tokyo, Japan) to remove molecules >10 kDa. Amino acid determination was performed as previously reported [15 (link),16 (link)]. Free amino acid concentrations were measured in μmol/dL.

5 g of cells were broken using a Retsch MM400 mill for 180 s at 30 Hz (repeat three times). The broken cells were then resuspended in lysis buffer (50 mM Hepes-KOH [pH 7.6], 100 mM KCl, 1 mM EGTA, 10% Glycerol, 0.1% NP40, 1 mM MgCl₂, 1 µg/mL leupeptin, 1 µg/mL pepstatin, 1 µg/mL chymostatin, and 1 mM Pefabloc) and then sonicated twice for 30 seconds on ice (1.2 W per mL). After centrifugation (5′, 4°C, 4000 rpm), the supernatant was filtered through a 2.7 µM then a 1.6 µM glass microfiber filter (Whatmann 25 mm GD/X). 25 µL of 50/50 slurry of anti-Flag M2 affinity agarose beads (Sigma) was added and incubated on a rotating wheel at 4°C for 30′. The beads were then washed three times in cold lysis buffer and then twice in 50 mM Hepes-KOH [pH 7.6], 50 mM KCl. Proteins were eluted by two 5′ incubations in 50 µL of 50 mM H3PO4.