RIPA buffer For 1000 ml 50 mM Tris HCl, PH 7.4 50 ml 150 mM NaCl 8.76 ml 1% Triton X-100 or NP-40 10 ml 0.5% Sodium deoxylcholate 5 g 0.1% SDS 1 g 1 mM EDTA (0.5M stock) 2 ml 10 mM Naf 0.42 g Add ddH2O to 1000 ml Add PMSF to a final concentration of 1mM and any...
In all, 24 h after transfection, cells were harvested and lysed in lysis buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100) containing a protease inhibitor cocktail. In total, 1000 μg of supernatants were incubated with Flag magnetic beads or protein G beads pre-coup...
For purification, the bacterial pellet was resuspended in the lysis buffer (50 mM Na2HPO4/NaH2PO4 (pH 8.0), 500 mM NaCl, 0.01 mM ZnCl2, 10% glycerol). Protein lysis was performed via SLM AMINCO French Press (Thermo Fisher Scientific), and the protein solution was centrifuged (40,000g, 45...
Approximately 1 × 106 cultured cells were harvested and lysed in RIPA lysis buffer (50 mM Tris–Cl pH7.5, 150 mM NaCl, 0.5% Triton X-100, 2 mM EDTA, 2 mM EGTA 25 mM NaF, 10% glycerol) containing 1 × protease inhibiters (Roche, Basel, Switzerland) for 30 min placed on...
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...
1981). Briefly, the leaf tissues of transgenic tobacco were cut into 2 mm square pieces using a sharp razor blade. The sliced leaf pieces were digested in 20 mL protoplast isolation buffer (0.25% Cellulase ONOZUKA R-10, 0.04% Pectinase, 400 mM sorbitol, 20 mM MES-KOH, pH 5.2, 0.5 mM...
As a reference one flow cell was activated and deactivated in the absence of any protein. The interaction experiments with four R subunit were performed at 25°C in running buffer (20 mM MOPS pH 7, 150 mM NaCl, 1 mM ATP, 5 mM MgCl2, 50 μM EDTA, 0.005% surfactant P20) at a flow...
After a 4-h incubation, cells were washed and incubated with surface antibodies for 30 min, and then cells were washed twice and incubated with fixation buffer for 2 h, followed by intracellular cytokines staining, which was performed for 45 min in permeabilization buffer. Flow cytometry ...
Cell lysates were collected by cell lysis in modified RIPA lysis buffer (20 mM TrisHCl pH 7.4, 5 mM MgCl2, 100 mM NaCl, 0.5% NP-40 and 1X Complete Protease Inhibitors) supplemented with 1 mM sodium orthovanadate, 1 mM AEBSF and 5 mM Levamisole. SDS-PAGE and western blot...
For a chymotrypsin assay, enzyme stock solution (1mg/mL, C4129, Merck) was utilized for standard dilutions (1:2, 1:5, 1:10, 1:50, 1:100, 1:250, 1:500) in a chymotrypsin buffer (0.532 g Tris-HCl, 0.198 g Tris, 2.925 g NaCl (0.05 M), and 0.735 g CaCl2 dihydrate (0.05 M...