Amicon ultra 4 50k

Saliva was eluted from sponges as described previously (69 (link)). For analysis of intestinal Abs, 2 to 3 g of freshly collected stool was added to 5 ml of sterile 1× phosphate-buffered saline (PBS) plus 0.5% bovine serum albumin supplemented with 50 μl of 100× protease inhibitor cocktail (Sigma-Aldrich, Corp., St. Louis, MO) and snap-frozen. Clarified fecal extracts were prepared as previously described (70 (link)) and concentrated to approximately 0.5 ml with an Amicon Ultra-4 50K centrifugal filter unit (Millipore, Billerica, MA).
1086.C gp120 IgG and IgA Ab levels in fecal extracts and saliva were measured by BAMA with gp120-conjugated Bio-Plex magnetic beads (Bio-Rad Laboratories, Inc., Hercules, CA) as previously described (71 (link)). Total IgG or IgA concentrations in secretions were measured by enzyme-linked immunosorbent assay (ELISA) with previously calibrated normal monkey serum as the reference standard (72 (link)). Anti-gp120 IgG or IgA concentrations measured in each secretion were divided by the total IgG or IgA concentration to obtain the specific activity (nanograms of IgG or IgA per microgram of total IgG/IgA). The specific activity was considered significant if it was greater than the mean activity measured in naive-infant control samples plus 3 standard deviations.

Yeast strains AJY2781 and AJY2846 were grown in 2L of YPD to OD600 of 1.0. Cells were transferred to ice for 1h before harvesting by centrifugation at 4°C. Cell pellets were washed and resuspended in 6ml of binding buffer (20mM HEPES-KOH, pH 7.6, 60mM NH₄Cl, 5mM Mg(oAc)₂, 2mM DTT, 1mM PMSF). Cells were disrupted by glass bead lysis and cleared by centrifugation at 30,000 g for 25 min at 4°C. Active 80S ribosomes were purified from cell extracts using cysteine-charged sulfolink columns as described [41 (link)]. To dissociate subunits, ribosome pellets were resuspended in 1ml elution buffer (20mM HEPES-KOH, pH 7.6, 60mM NH₄Cl, 500mM KCl, 10mM Mg(oAc)₂, 2mM DTT, 1mM PMSF and 1uM each leupeptin and pepstatin) and incubated at 37°C for 30 min after the addition of puromycin and GTP, each to 1mM final concentration. The sample was then centrifuged through 10–30% sucrose gradients in elution buffer for 4h at 32,000 rpm in an SW40 rotor (Beckman Coulter). Fractions containing the 60S and 40S peaks were pooled separately and concentrated using an Amicon Ultra-4 50 K (Millipore), and buffer was changed to Ribosome storage buffer (20mM HEPES-KOH, pH 7.6, 100mM KCl, 5mM Mg(oAc)₂, 2mM DTT, 250mM sucrose).