Graphy using the Multiple Affinity Removal Column (MARS Hu-14, Agilent Technologies > 자유게시판

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Graphy using the Multiple Affinity Removal Column (MARS Hu-14, Agilent Technologies). This technique has proven to be an interesting tool in the search for plasma biomarkers [32,35,36]. Depletion was performed by high performance liquid chromatography (HPLC) using a 1200 series HPLC chromatograph (Agilent Technologies) equipped with a manual injector (9725i, Agilent Technologies). After PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/11719833 several chromatographic cycles, aliquots of the flow-through fractions containing low-abundance proteins were combined and desalted using centrifugal filter devices with a 3 kDa cut off (Amicon Ultra, Millipore). These samples were stored at -80 prior to analysis and the protein concentration was determined using the Bradford-Lowry method [37].Two-dimensional differential in gel electrophoresis (2D-DIGE)protein). Protein extracts were diluted in rehydration buffer (30 mM TRIS, 7 M Urea, 2 M Thiourea, 4 CHAPS) and applied to 24 cm pH 4? IPG strips. The first dimension was run on the IPGphor IEF II System (GE Healthcare) in darkness, as follows: 500 V for 30 minutes, a linear gradient to 3500 V over 3 h, 3500 V for 3 h, a linear gradient to 6000 V over 3 h, and 6000 V until 69000 v/h. After the first dimension, the strips were equilibrated in SDS-equilibration buffer (1.5 M Tris/HCl [pH 8.8], 6 M Urea, 87 Glycerol and 2 SDS). To increase the reproducibility an electrophoresis system "Ettan DALTsix" (GE Healthcare) was used for the polymerization of the gels. To run the second dimension 10 acrylamide gels were used. To do this, 1.5 M Tris?HCl pH 8.8 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/13867361 was used, 10 SDS (w/v) and Methyl 5-amino-2,4-difluorobenzoate milliQ water were used to obtain the desired concentration of acrylamide. APS and TEMED (BioRad) were also added to catalyst the polymerization reaction. After preparing the gels, each IPG strip was placed horizontally on the top of the gel, avoiding bubbles to remain trapped between the strip and the surface of the gel. Electrophoresis took place at 25 , firstly applying 5 W/gel for 20 min and then 1 W/gel overnight in the presence of running buffer (25 mM Tris, 0.2 M glycine, 3 mM SDS).Image acquisition and analysisNSTEACS plasma samples (n = 5) and healthy control samples (n = 5) or STEACS plasma samples (n = 6) and healthy control samples (n = 6) with similar clinical parameters were used in both 2D-DIGE analyses. This technique is based on the specific linkage of three fluorochromes with differential spectral properties (Cy2, Cy3 and Cy5). It permits the separation of multiple samples in a single gel increasing the reproducibility and reliability of the 6-Bromopyridine-2,3-diamine analysis of differential expressed proteins. Proteins were labelled according to the manufacturer's instructions (GE Healthcare), such that 50 g of depleted plasma proteins from NSTEACS or STEACS patients and healthy controls were labeled with 400 pmol of N-hydroxysuccinimide Cy3 or Cy5 fluorescent cyanine dye and the labeling reaction was then quenched with 0.2 mM lysine. All experiments included an internal standard containing equal amounts of each protein extract labeled with 400 pmol of N-hydroxysuccinimide Cy2 dye. The internal standard and two additional plasma samples (NSTEACS/STEACS and control) were combined and run on a single gel (150 g of totalAfter SDS-PAGE, the gels were scanned on a Typhoon TRIO fluorescence gel scanner (GE Healthcare) using appropriate individual excitation and emission wavelengths, filters and photomultiplier values sensitive for each of the Cy3, Cy5 and Cy2 dyes. Relative protein quantificat.