Analysis of Normal and Neoplastic Tissue NHC Proteins by High-Resolution Two-Dimensional Gradient Electrophoresis and Silver Staining
Isaac Bekhor, Carol J. Mirell, C. C. Liew in Progress in Nonhistone Protein Research, 1985
During initial preliminary studies, we utilized Coomassie brilliant blue R-250 for the visualization of proteins. This dye can detectably stain 0.1 to 3.0 μg of protein in a single band in slab polyacrylamide SDS gels. Since in single-dimensional protein gel displays a band in a rod or slab may in fact represent several “spots” in a two-dimensional gel profile, many NHC proteins present in low concentration in samples are not detected by Coomassie. We now routinely use silver stain and in vitro labeling of proteins by reductive methylation for protein visualization.43,96 In our hands, silver “stain” detects proteins some 370-fold more sensitively than Coomassie, this sensitivity being in the same range as that for 14C and 3H-methylated proteins after a 5-day autoradiographic exposure. Silver staining utilizes a modification of de Olmos’ cupric-silver method and possesses limits of sensitivity in our gel system of less than 3 pg/mm3.43
Mechanisms of Fibril Formation and Cellular Response
Martha Skinner, John L. Berk, Lawreen H. Connors, David C. Seldin in XIth International Symposium on Amyloidosis, 2007
37 °C for 1 h. CD spectra were obtained by using a JASCO J-720 spectropolarimeter at 25 °C. A molecular mass of the TTR of 14 kDa was used for calculation of the mean residue ellipticity. The fluorescence intensity of Trp was measured via a Hitachi F-4500 spectrofluorimeter at 25 °C. All assays used excitation at 295 nm and emission at 340 nm. Excitation and emission slits were set at 5 nm. To assess the amount of amyloid fibrils in vitro, thioflavin T test has been widely used. Fluorescence spectra were obtained by using a HITACHI F-4500 spectrofluorimeter with an assay volume of 1 ml. All assays used excitation at 450 nm and emission at 482 nm. Excitation and emission slits were set at 5 nm. The reaction mixture contained 5 uM thioflavin T and 50 mM Gly-NaOH buffer, pH 10.0. Nonboiled SDS-PAGE was performed under nondenaturing conditions. One microgram of the TTR samples incubated for 37 °C for 5 days at pH 3.0 as described above was neutralized with PBS to obtain a final pH greater than 6.5. After neutralization, samples were mixed with 5% SDS sample buffer and loaded on 15% polyacrylamide gels, which were stained with Coomassie Brilliant Blue. Intensities of the bands were evaluated by densitometric analysis using ATTO densito.
Membrane Proteins Involved in the Water Permeability of Human Erythrocytes. Binding of p-Chloromercuribenzene Sulfonate to Membrane Proteins Correlated with Nuclear Magnetic Resonance Measurements
Gheorghe Benga in Water Transport in Biological Membranes, 1989
During PAGE, β-mercaptoethanol was omitted and 20 mM N-ethylmaleimide (NEM) was added to prevented the release of PCMBS and its subsequent binding to NEM binding sites. Membrane peptides were separated using the discontinuous SDS polyacrylamide gel system designed by Laemmli,43 as previously described.44 The slab gels used consisted of a running gel of 7.5% acrylamide and 5% stacking gel. The acrylamide to bis-acrylamide ratio was maintained at 36.5:1 in both the stacking and running gel. The slab gels were 16 cm × 14 cm × 0.1 cm. Running times were 1 hr at 70 V and then 3 hr at 125 V (18°C) in buffer (25 mM Tris, 192 mM glycine, and 0.1% SDS). Following electrophoresis, gels were cut into 2-mm slices and the radioactivity measured with the gammacounter. Parallel samples were stained overnight with a solution containing 0.075% Coomassie Brilliant Blue R 250, 45% methanol, and 10% acetic acid. Destaining was performed with 10% acetic acid followed by drying as previously described.45 The proportion of various membrane peptides was calculated from densitometric scans obtained using a microdensitometer, MD 100, coupled to an automatic integrating recorder, K 201 (both manufactured by C. Zeiss, Jena, GDR).
Effect of poly(ethylene glycol) content and formulation parameters on particulate properties and intraperitoneal delivery of insulin from PLGA nanoparticles prepared using the double-emulsion evaporation procedure
Published in Pharmaceutical Development and Technology, 2018
Yusuf A. Haggag, Ahmed M. Faheem, Murtaza M. Tambuwala, Mohamed A. Osman, Sanaa A. El-Gizawy, Barry O’Hagan, Nigel Irwin, Paul A. McCarron
SDS-PAGE analysis was performed using a BioRad Mini Protean II gel apparatus (Hercules, CA). The final supernatant obtained after 7 days of the in vitro release experiment was used as a sample for this study. The sample was prepared under non-reducing conditions for application on a NuPAGE® gel consisting of 4% and 12% stacking and resolving gels, respectively. A fixative solution of Coomassie Brilliant Blue was employed to stain and reveal the protein bands. Insulin dispersed in PBS (pH 7.4) was used as control to simulate release conditions. Electrophoresis was run in constant current mode (50 mA) and fixed voltage modes (60 and 120 V) during stacking and running stages, respectively (Park et al. 1998). The GelDoc-It™ image system was used to record the position of protein bands.
Cytotoxic, necrotic, apoptotic, and autophagic properties of venom sac extract of Vespa orientalis in T47D and MCF10A breast cell lines
Published in Toxin Reviews, 2023
Seyed Mohammad-Hossein Shetab-Boushehri, Asieh Hosseini, Javad Rafinejad, Alireza Ebadollahi-Natanzi, Seyed Vahid Shetab-Boushehri
The protein content of the VSE of hornet was measured according to modified Bradford protein assay (Zor and Selinger 1996). Briefly, a 50 ml of 0.1 mg.ml−1 solution of coomassie brilliant blue G-250 in a mixture of 95% ethanol (2.5 ml), 85% orthophosphoric acid (5 ml), and distilled water (42.5 ml) was made and used as a reagent. A 200 µg.ml−1 stock solution of BSA in DW was made and serially diluted to make 10, 20, 30, 50, 70, 90, 100, 120, 140, 160, 180, and 200 µg.ml−1 working solutions of BSA in DW. To 5 µl of each standard protein solution or 100-fold diluted sample of VSE, 250 µl of coomassie brilliant blue reagent was added in each well of a 96-well plate. After 5 min, absorbances of solutions were measured at 450 nm and 590 nm by a multi-mode microplate reader (Synergy™ HT Microplate Reader, BioTek Instruments Inc., Winooski, Vermont, USA). The absorbance of DW as blank was subtracted from those of standards and the VSE sample. Subtracted absorbances of standards and VSE sample at 590 nm were then divided by respective absorbances at 450 nm. The protein standard (calibration) curve was constructed by depicting the 590 nm/450 nm absorption ratio of standards versus their different concentrations. Protein concentration of VSE was determined by interpolation of 590 nm/450 nm absorption ratio of VSE sample over tabulated standards concentrations on protein standard curve followed by multiplication by the dilution factor. The measurements were performed in triplicate samples.
Cholesterol-linoleic acid liposomes induced extracellular vesicles secretion from immortalized adipose-derived mesenchymal stem cells for in vitro cell migration
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2023
Jzit Weii Chen, Fong Fong Liew, Hsiao Wei Tan, Misni Misran, Ivy Chung
Acetic acid, chloroform and hydrochloric acid were obtained from R&M Chemicals, Kuala Lumpur, Malaysia. Low-molecular-weight chitosan and cholesterol were obtained from Sigma-Aldrich (St. Louis, MO). Linoleic acid and DOTAP were obtained from Fluka (Buchs, Switzerland) and Avanti Polar Lipids (Alabaster, AL), respectively. Analytical grade sodium nitrate was obtained from HmbG Chemicals (Kuala Lumpur, Malaysia). Sodium hydroxide (reagent grade, ≥98%, pellets (anhydrous)) was obtained from Merck (Solna, Sweden). Low glucose Dulbecco’s modified Eagle medium (DMEM), foetal bovine serum (FBS), penicillin–streptomycin (Pen–strep) (10,000 U/ml) and GlutaMax were obtained from Gibco (Carlsbad, CA). Immortalized AD-MSCs, ASC52telo were obtained from America Type Culture Collection (ATCC) (Manassas, VA). Human keratinocytes (HaCaT) cell line and optimized DMEM were obtained from AddexBio (San Diego, CA). Pierce™ bovine serum albumin (BSA) standard (2 mg/ml) was obtained from Thermo Fisher Scientific (Waltham, MA). Cell culture falcon tubes, dishes and plates were obtained from NEST Biotechnology Co. (Wuxi, China). Dulbecco’s phosphate-buffered saline (DPBS) was obtained from Sangon Biotechnology (Shanghai, China). Minisart® syringe filter, surfactant-free cellulose acetate (SFCA) and pore size of 0.8 µm were obtained from Sartorius (Goettingen, Germany). Amicon® Ultra-15 centrifugal filter devices-10 kDa and exoEasy Maxi Kit were obtained from Merck (Darmstadt, Germany) and Qiagen (Hilden, Germany). Coomassie Brilliant Blue (CBB) dye was obtained from Nacalai Tesque Inc. (Kyoto, Japan).
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