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- W2075667709 abstract "Proteomic profiling of mouse liver microsomes was performed by SDSpolyacrylamide gel electrophoresis (1D-SDS-PAGE) and nano-liquid chromatography tandem mass spectrometry (nano-LC-MS/MS) for the pilot study of Membrane Proteome Initiative (MPI) of AOHUPO.More than one hundred proteins were identified with high MS/MS search score, and most of those proteins have been reported to be membrane associated proteins such as microsomal glutathione S-transferase 1, cytochrome P-450 and low density lipoprotein receptor-related proteins. This platform was used for ER membrane proteomics of Jurkat cells of a human lymphoblatic lymphoma cell line to find marker proteins for heat stress. The microsomes were prepared by two-step centrifugation of liver homogenate, firstly the homogenate was centrifuged at 15,000 g for 15 min to yield supernatant and secondly the supernatant was centrifuged at 132,000 g for 60 min to yield microsomes pellet. The microsome pellet was suspended in 0.1 M sodium carbonate solution containing 0.5 mM PMSF and 10 ug/mL of aprotinin and leupeptin to yield highly purified microsomes in the pellet by centrifugation at 132,000 g for 60 min. The purified microsomes were pretreated with ice cold acetone-methanol (8:1) solution (Anal.Biochem., 273, 313-315, 1999) to be applied for the separation of membrane proteins by 1D-SDS-PAGE with a gradient gel (5-20%) to yield the fine separation of proteins in more than 50 bands which were visualized by Coomassie Blue staining. The protein bands were cut out and submitted to in gel doi:10.4172/jpb.s1000091" @default.
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- W2075667709 date "2008-07-01" @default.
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- W2075667709 title "1D-SDS-PAGE and Nano-LC-MS/MS for Membrane Proteomics of Mouse Liver Microsomes (MPI sample) and its Application to Human Proteomics of ER from Jurkat Cells" @default.
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