Briefly, active fractions were reduced by incubation at 57˚C for 30 min with 10 mM 1,4-dithiothreitol (DTT) and 10 mM ammonium bicarbonate buffer, alkylated with 50 mM of indole-3-acetic acid (IAA) at room temperature for 45 min without light access, and then digested overnight at ...
Buffer A consisted of 1% (v/v) Triethylammonium bicarbonate (TEAB) in water and buffer B of 80% (v/v) ACN and 1% (v/v) TEAB in water. The sample was loaded onto the column at 2 μL/min at a maximum 500 bar, fractionated using the following gradient: 3% to 40% B in 57...
The sample was diluted to 0.6 M final Gn-HCl concentration with 25 mM ammonium bicarbonate buffer. For digestion of protein, trypsin was added in a trypsin-to-lysate ratio of 1:50 after and incubation was performed overnight at 37 °C. The supernatant was vacuum dried and the peptides ...
aAmmonia react with water and carbon dioxide to form a molecule of ammonium bicarbonate, this is the first step. The second step is: ammonium bicarbonate and sodium chloride precipitation reactions to generate a molecule of ammonium chloride and sodium bicarbonate, sodium bicarbonate precipitation becau...
Cells were lysed on ice in buffer containing 50 mM HEPES (pH 7.5), 0.5% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM sodium fluoride, 2.5 mM sodium orthovanadate, 1X protease inhibitor cocktail (Roche, Basel, Switzerland), and 1% each of phosphatase inhibitor cocktails 2...
Osteopetrosis is a skeletal disorder of humans and animals characterized by the formation of overly dense bones, resulting from a deficiency in the number and/or function of bone-resorbing osteoclast cells. In cattle, osteopetrosis can either be induced during gestation by viral infection of the dam...
The concentration of the samples was normalized using 50 mm ammonium bicarbonate to yield a final concentration of 1 μg/μl. Approximately 100 μg of proteins from each sample was subjected to in-solution trypsin digestion to generate peptides. Disulfide bonds were reduced by incubation of ...
Parasitized blood from these animals was diluted in Krebs-Ringer bicarbonate buffer (Sigma-Aldrich, St. Louis, MO) and normal saline to deliver 105 iRBCs i.p. in 200 μl into experimental WT or IL-10 KO mice. Thin blood smears were collected at regular intervals to monitor for peripheral...
Supernatant was collected and protein assays were performed using a Pierce BCA Protein Assay kit (Thermo Fisher Scientific). Fifteen to 20 μg of total protein—or 0.5–5E + 9 particles (p)—were resuspended in a 4 × LDS buffer (Thermo Fisher Scientific) and a 10 × sample ...
Triple-negative breast cancer (TNBC) has a poor clinical outcome, due to a lack of actionable therapeutic targets. Herein we define lysosomal acid lipase A (LIPA) as a viable molecular target in TNBC and identify a stereospecific small molecule (ERX-41) that binds LIPA. ERX-41 induces endopl...