| 11. |
- Edvardsson, Louise, et al.
(författare)
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Real-time PCR analysis for blood cell lineage specific markers.
- 2009
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1940-6029. ; 496, s. 313-322
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Tidskriftsartikel (refereegranskat)abstract
- We here describe the methods for the isolation of distinct hematopoietic subpopulations, as defined by their immune phenotype by fluorescence-activated cell sorting, and how these cells can be analyzed even at a single-cell level for the gene expression of a number of transcription factors and other differentiation markers.
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| 12. |
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| 13. |
- Everberg, Henrik, et al.
(författare)
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Enrichment of membrane proteins by partitioning in detergent/polymer aqueous two-phase systems.
- 2008
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1940-6029. ; 424, s. 403-412
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Tidskriftsartikel (refereegranskat)abstract
- Methods that combine efficient solubilization with enrichment of proteins and intact protein complexes are of central interest in current membrane proteomics. We have developed methods based on nondenaturing detergent extraction of yeast mitochondrial membrane proteins followed by enrichment of hydrophobic proteins in aqueous two-phase system. Combining the zwitterionic detergent Zwittergent 3-10 and the nonionic detergent Triton X-114 results in a complementary solubilization of proteins, which is similar to that of the anionic detergent sodium dodecyl sulfate (SDS) but with the important advantage of being nondenaturing. Detergent/polymer two-phase system partitioning offers removal of soluble proteins that can be further improved by manipulation of the driving forces governing protein distribution between the phases. Integral and peripheral membrane protein subunits from intact membrane protein complexes partition to the detergent phase while soluble proteins are found in the polymer phase. An optimized solubilization protocol is presented in combination with detergent/polymer two-phase partitioning as a mild and efficient method for initial enrichment of membrane proteins and membrane protein complexes in proteomic studies.
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| 14. |
- Everberg, Henrik, et al.
(författare)
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Extraction of yeast mitochondrial membrane proteins by solubilization and detergent/polymer aqueous two-phase partitioning.
- 2009
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1940-6029. ; 528, s. 72-81
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Tidskriftsartikel (refereegranskat)abstract
- Identification and characterization of membrane proteins is of increasing importance in modern proteomic studies. It is of central interest to have access to methods that combine efficient solubilization with enrichment of proteins and intact protein complexes. Separation methods have been developed based on nondenaturing detergent extraction of yeast mitochondrial membrane proteins followed by enrichment of hydrophobic proteins in aqueous two-phase system. Combining the zwitterionic detergent Zwittergent 3-10 and the nonionic detergent Triton X-114 results in a complementary solubilization of proteins, which is similar to that of the anionic detergent sodium dodecyl sulfate (SDS) but with the important advantage of being nondenaturing. Detergent/polymer two-phase system partitioning offers removal of soluble proteins, which can be further improved by manipulation of the driving forces governing protein distribution between the phases. Integral and peripheral membrane protein subunits from intact membrane protein complexes partition to the detergent phase while soluble proteins are found in the polymer phase. A protocol is presented which combines nondenaturing solubilization of membrane proteins with extraction in detergent/polymer two-phase system for application in proteomic studies as a mild and efficient method for enrichment of membrane proteins and membrane protein complexes.
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| 15. |
- Galaev, Igor, et al.
(författare)
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Affinity processing of cell-containing feeds using monolithic macroporous hydrogels, cryogels.
- 2008
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1940-6029. ; 421, s. 247-255
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Tidskriftsartikel (refereegranskat)abstract
- Monolithic macroporous hydrogels, "cryogels," are produced by polymerization in a partially frozen state when the ice crystals perform as a porogen. Cryogels have a unique combination of properties: (i) large (10-100 microm) pores; (ii) minimal non-specific interactions due to the hydrophilic nature of the polymers; (iii) porosities exceeding 80-90%; (iv) good mechanical stability. These properties of cryogels allow for their application for direct capture of extracellularly expressed histidine-tagged protein from the fermentation broth and separation of different cell types.
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| 16. |
- Gussing, Fredrik, et al.
(författare)
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Selective protein expression within the CNS using hybrid lentivirus.
- 2009
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1940-6029. ; 515, s. 215-226
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Tidskriftsartikel (refereegranskat)abstract
- Lentiviral vectors offer many advantages within the central nervous system. They are capable of transfecting neurons and can be readily manipulated for selective expression. This chapter describes the production and use of lentiviruses to deliver GFP to the central nervous system.
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| 17. |
- Gärdén, Per, et al.
(författare)
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Proteomic data exchange and storage: using proteios
- 2007
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Ingår i: Methods in Molecular Biology. - 1940-6029. ; 367, s. 241-260
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Tidskriftsartikel (refereegranskat)abstract
- Proteios (http://www.proteios.org) is an initiative for the development of a comprehensive open source system for storage, organization, analysis, and annotation of proteomics experiments. The Proteios platform is based on existing principles for proteomics data publishing and data exchange.
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| 18. |
- Liu, Hanguan, et al.
(författare)
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Identification of promoter elements in 5'-flanking region of murine cyclic nucleotide phosphodiesterase 3B gene
- 2005
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Ingår i: Phosphodiesterase Methods and Protocols (Methods in molecular biology). - New Jersey : Humana Press. - 1064-3745 .- 1940-6029. ; 307, s. 109-124
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- We describe techniques for identifying functional promoter elements in the 5'-flanking region of the murine cyclic nucleotide phosphodiesterase 3B (mPDE3B) gene. The 5'-flanking region of the mPDE3B gene was cloned and sequenced, and putative transcription factor binding sites were identified with computational tools. A series of reporter plasmids containing the luciferase gene fused to different fragments of the 5'-flanking region of the mPDE3B gene was constructed and used to transfect 3T3-L1 fibroblasts or differentiating adipocytes. Reporter gene assays showed that there are two promoter regions in the 5'-flanking region in the mPDE3B gene: a distal region located approx 4 kb upstream of the translation initiation site that contains cAMP-response element (CRE) cis-acting elements, and a proximal region that is GC rich and lacks TATA sequences. The distal promoter region induced much higher luciferase activity than did the proximal one. Mutation of the CRE sequences or reversal of the orientation of the CRE-containing region abolished promoter activity of the distal region. Electrophoretic mobility shift assay analysis indicated that binding to CRE elements was greater in nuclear extracts from differentiating adipocytes than from fibroblasts. Mapping of transcription initiation sites suggested that the distal promoter region might function as an enhancer, whereas the proximal promoter drives transcription of the mPDE3B gene.
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| 19. |
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| 20. |
- Mattila, Ismo, et al.
(författare)
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Application of lipidomics and metabolomics to the study of adipose tissue
- 2008
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1064-3745 .- 1940-6029. ; 456, s. 123-130
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Tidskriftsartikel (refereegranskat)abstract
- Role of specific reactive lipids as well as amino acids in control of insulin signalling in adipose tissue is well recognized. Since it is practically impossible to measure the levels of all metabolites in the biological sample simultaneously with a single analytical platform, we utilize multiple platforms to study the lipids and metabolites of relevance to adipose tissue metabolism and insulin signalling. Two screening platforms cover a broad range of lipid molecular species (UPLC/MS based lipidomics platform) as well as organic acids and sterols (GCxGC-TOF platform). A targeted platform for amino acids (UPLC) is also applied.
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