PDF Publication Title:
Text from PDF Page: 016
media for 1min at RT. After incubation, cells were washed several times with PBS and immediately analyzed by fluorescence microscopy using the appropriate filter system. Emission Spectrometry, ICP-AES (Optima 3100, Perkin Western blotting analysis SKBR3 cells were treated for 24h with IC50 concentrations of AgNPs-EPS and with 200 μg/ml of free- metal EPS. After washing with PBS, cells were carefully scraped and incubated on ice for 30 min Lysis buffer containing 7 M urea, 2 mM Thiourea, 0.4 % w/v CHAPS, 1 % w/v 1,4-dithioerythritol (DTE) and 30mM TRIZMA base, pH 8.5. The total cellular lysate was centrifuged at 14,000 rpm for 10 min to clear cell debris and protein concentration determined by Bradford assay, as already reported [75, 76]. Protein samples (20μg/lane) were subjected to SDS polyacrylamide gel electrophoresis, then transferred to a nitrocellulose membrane (HyBond ECL, Amersham) and stained with Ponceau S (Sigma Aldrich). Western blotting analyses were performed using a rabbit polyclonal antibodies for Akt 1-2-3 and pAkt 1-2-3, a goat polyclonal antibody for Beclin 1, a mouse monoclonal antibody for Actin β, HSP90, MMP2 and MMP9 by Santa Cruz Biotechnology (Santa Cruz, CA, USA), a rabbit polyclonal antibody for LC3 by Sigma Aldrich and a rabbit polyclonal antibodies for p62, ATG5 and ATG7 by Cell Signaling Technology. Following incubation with the appropriate peroxidase-linked antibody, the reaction was revealed by the ECL detection system, using high performance films (Hyperfilm ECL, Amersham), as already described [68, 77]. The correct protein loading was ascertained by red Ponceau staining and immunoblotting for Actin β. Subcellular separation for Ag+ release After treatments, SKBR3 cells were detached from the flasks with a scaper and resuspended in Sucrose buffer (5mM Tris-HCl pH 7.4, 0.32M Sucrose and 0.001 mg/ mL Protease/phosphatase inhibitor). Nuclei were pelleted by centrifugation at 1000 g for 20 minutes; mitochondria from the post-nuclear supernatants were recovered by centrifugation at 8500 g for 30 minutes; lysosome by centrifugation at 20000 g for 30 minutes and membranes by centrifugation at 105000 g for 90 minutes. The Ag+ release from subcellular components was measured by scanning electrochemical microscopy and anodic stripping voltammetry (ASV) using a CHI920B workstation (CH Instruments), as already described [24, 25]. Determination of total Ag Total silver was determined in 1 mg sample of pulverized AgNPs-EPSaer, AgNPs-EPSanaer and in subcellular fractions of breast cancer cell line SKBR3. Elmer) [78]. 2D-DIGE analysis Protein samples were labeled for 2D-DIGE analysis using CyDyeTM DIGE minimal labeling kit (GE Healthcare, Sweden), according to manufacturer’s recommendations as already described [18]. An internal standard was generated by combining equal amounts of samples and labeled with Cy2 Protein samples (50 μg) were labeled with 200 pmol of CyDye on ice in the dark for 30 min. Labeling reaction was quenced by addition of 1 μl of 10 mM lysine, and incubation was continued on ice for 10 min, in the dark. The first dimensional separation was performed at 20°C on commercial sigmoidal immobilized pH gradient strips (IPG), 18 cm long with pH range 3.5 to 10 (Pharmacia). Strips were rehydrated in 8 M urea, 2% CHAPS, 10 mM DTE, and 0.5% carrier ampholytes (Resolyte 3.5–10). The isoelectrofocusing was carried out by linearly increasing voltage from 200 to 3500 V during the first 3 hr, after which focusing was continued at 8000 V for 8 hr, as already described [33, 68–72, 75–77, 79–84]. After the first dimension separation the IPG strips were equilibrated with a solution containing 6 M urea, 30% glycerol, 2% SDS, 0.05M Tris-HCl pH 6.8, and 2% DTE for 12 min, to resolubilize proteins and reduce disulphuric bonds. The -SH groups were then blocked by substituting the DTE with 2.5% iodoacetamide in the equilibrating buffer. The focused proteins were then separated on 9–16% linear gradient polyacrylamide gels (SDS- PAGE) using a DALT six (GE Healthcare) apparatus with a constant current of 40 mA/gel at 10°C. Images were acquired with a Typhoon FLA 9500 scanner (GE Healthcare), using specific emission filters, and analyzed by the Image Master 2D Platinum 7 software (GE Healthcare). Protein spots showing more than 1.3 fold change in spot volume (increased for up- regulation or decreased for down-regulation), with a statistically significant ANOVA value (p ≤ 0.05), were considered differentially represented and further identified by MS analysis. After the acquisition, each gels were stained with ammoniacal silver nitrate, as already described [80]. In-gel digestion and MS analysis of tryptic digests Spots of interest were manually picked and mass spectrometric sequencing was carried out after in-gel digestion, using sequencing-grade trypsin (20 μg/vial), Samples were digested with 2 mL of aqua regia, heating at 60° until a solution was obtained. Total Ag was determined by Inductively Coupled Plasma Atomic www.impactjournals.com/oncotarget 9700 OncotargetPDF Image | Anticancer activity of biogenerated silver nanoparticles
PDF Search Title:
Anticancer activity of biogenerated silver nanoparticlesOriginal File Name Searched:
oncotarget-09-9685.pdfDIY PDF Search: Google It | Yahoo | Bing
Turbine and System Plans CAD CAM: Special for this month, any plans are $10,000 for complete Cad/Cam blueprints. License is for one build. Try before you buy a production license. More Info
Waste Heat Power Technology: Organic Rankine Cycle uses waste heat to make electricity, shaft horsepower and cooling. More Info
All Turbine and System Products: Infinity Turbine ORD systems, turbine generator sets, build plans and more to use your waste heat from 30C to 100C. More Info
CO2 Phase Change Demonstrator: CO2 goes supercritical at 30 C. This is a experimental platform which you can use to demonstrate phase change with low heat. Includes integration area for small CO2 turbine, static generator, and more. This can also be used for a GTL Gas to Liquids experimental platform. More Info
Introducing the Infinity Turbine Products Infinity Turbine develops and builds systems for making power from waste heat. It also is working on innovative strategies for storing, making, and deploying energy. More Info
Need Strategy? Use our Consulting and analyst services Infinity Turbine LLC is pleased to announce its consulting and analyst services. We have worked in the renewable energy industry as a researcher, developing sales and markets, along with may inventions and innovations. More Info
Made in USA with Global Energy Millennial Web Engine These pages were made with the Global Energy Web PDF Engine using Filemaker (Claris) software.
Infinity Turbine Developing Spinning Disc Reactor SDR or Spinning Disc Reactors reduce processing time for liquid production of Silver Nanoparticles.
| CONTACT TEL: 608-238-6001 Email: greg@infinityturbine.com | RSS | AMP |