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1. The buffer system The pH of the solution is critical. Proteins may precipitate or become unstable when the pH is outside of the physiological range. To avoid this situation, a buffer system such as Tris-HCl is recommended. Besides buffering solutions in this range, a Tris-HCl buffer pre...
Define the term radiation buffer used in chemistry. Is a solution of 0.05 M NH3 and 0.02 M HCl a buffer? Explain why or why not. Explain how a buffer is able to resist changes in pH. What is the best buffer? What are the rules of reasoning? Define the term "buffer solution" and...
For affinity-precipitation, proteins (~5 mg) were solubilized from five dishes (round, 10 cm in diameter) of the heavy- and light-labeled cells, respectively, with a lysis buffer containing 2% dodecyl maltoside in 150 mM NaCl and 20 mM Tris-HCl (pH 7.4). The solubilized proteins...
The culture was incubated for 20h at 18C, bac- teria were harvested by centrifugation (6000 g, 10 min, 4C) and resuspended in lysis buffer (50 mM Tris/HCl at pH 8, 150 mM NaCl, 2 mM b-mercaptoethanol). After sonication and rem...
Cells were collected, lysed and sonicated at 4 °C in a lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% SDS, 5 mM sodium pyrophosphate, protease and phosphatase inhibitor tablet). Cell lysates were centrifuged at 13,000g for 15 min, and the supernatant was recove...
The bacterial pellet was resuspended in 100 ml of lysis buffer (50 mM Tris-HCl pH 7.4, 50 mM NaCl, 10 mM imidazole, 10 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride [PMSF]), incubated with lysozyme (50 µg/ml; Roth) for 30 min in a cold room, and then broken in an...
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centrifugation and lysed in a lysis buffer (50 mM Tris-HCl, pH 7.4, 200 mM NaCl, 5% glycerol, 1 mM EDTA, 14 mM 2-mercaptoethanol, and 1 mM PMSF) using a sonicator. The recombinant protein was purified by affinity chromatography using Streptactin Beads 4FF (Smart-Lifesciences)...
For identifying the interacting partners of TM9SF4, A2780 and NIH3T3 cells, with or without GFP-TM9SF4 overexpression, were lysed in lysis buffer (50 mM Tris-HCl, pH 7.5; 150 Mm NaCl; 1% NP-40; 0.5% sodium deoxycholate; 0.2% Triton X-100 and protease inhibitor cocktail) at 4 ...