Then, pelleted cells were sonicated in RIPA buffer (150- mM NaCl, 50-mM Tris–HCl pH7.4, 0.5% (w/v) sodium deoxycholate, 1.0% (v/v) Nonidet P-40, 0.1% (w/v) SDS, 5-mM EDTA, 50-mM NaF) supplemented with 1 × complete protease inhibitors (Roche). A similar procedure was...
Particularly, the spacer group in the shape of a particle is suitable for use as a ligand immobilization support. The polyoxyethylene chain improves dispersability in water or a buffer solution and improves the handling property. If Q represents a linking group represented by the formula (a-1),...
For immunoblotting, cells were lysed in buffer A (20 mM HEPES, pH 7.4, 0.5% Triton X-100, 0.5% CHAPS, and 10% glycerol) or alternatively buffer B (0.05 M Tris-HCl. pH 8.0, 0.15 M NaCl, 5.0 mM EDTA, and 1% NP-40) with protease and phosphatase inhibitor cocktail ...
The cells were collected at each indicated time point following stimulation. The cells were washed with ice-cold phosphate-buffered saline (PBS) twice, and lysed on ice in lysis buffer (20 mM Tris-HCl pH 7.6, 150 mM NaCl, 2 mM EDTA, 0.5% NP-40) with a protease inhibitor cocktai...
40 μl of each cell lysate was incubated in a 96-well plate with 200 μl of caspase assay buffer (20 mM HEPES at pH7.5, 10% glycerol, 1 mM dithiothreitol) and caspase-3 fluorogenic substrate Ac-DEVD-AMC (BioMol Research Labs) at 37°C for 1 hour. Cleavage of this tetrapeptide ...
Purified phage was dialyzed using a Pierce 10 K Slide-A-Lyzer cassette (Thermo Fisher Scientific, Rockford, IL, USA) against 10 mM Tris-0.1 mM EDTA (pH 8.0) (TE) buffer to remove CsCl. Purified phage was stored at 4°C. Electron microscopy Purified phages were sedimented by centrifugation...
The purified ROS were washed three times with 50 ml isotonic buffer (10 mM Hepes pH 7.5, 100 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA, and 1 mM DTT) and PDE6 was isolated with three washes (50 ml) of hypotonic buffer (10 mM Hepes pH 7.5, 0.1 mM EDTA, 1 mM DTT). PDE6 was ...
EM. Mice were perfused via the heart with a buffered mix of 2.5% glutaraldehyde and 2% paraformaldehyde in phosphate buffer (0.1 M, pH 7.4). The animal was left for 2 h and then 100 mm thick sections cut in the transverse plane through the middle portion of the uterus using a vibratome...
The filters carrying the cells were then placed over filters presoaked with Z-buffer-X-Gal solu- tion according to the protocol of the Matchmaker system. The pBridge vector (Clontech) was used for yeast three-hybrid screening. The IDD14α cDNA was amplified by RT–PCR, and the PCR product...
121-130) with the following minor modifications: (1) Iodoacetamide (1 mM) was included in the homogenization buffer; (2) Homogenization was as published but for a shorter period of 10 seconds once and at a slower speed (setting 10); and (3) The equilibration step with KCl/EDTA was not...