| 341. |
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| 342. |
- Persson, Andrea, et al.
(author)
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Production and HPLC-Based Disaccharide Analysis of Xyloside-Primed Glycosaminoglycans
- 2022
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In: Glycosaminoglycans. Methods in Molecular Biology, vol 2303. Balagurunathan K., Nakato H., Desai U., Saijoh Y. (eds). - New York, NY : Springer. - 1064-3745 .- 1940-6029. - 9781071613986 ; , s. 173-182
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Book chapter (other academic/artistic)abstract
- Although glycosaminoglycans (GAGs) are known to be involved in a variety of physiological and pathological processes, knowledge about their expression by cells or tissues, the GAGome, is limited. Xylosides can be used to induce the formation of GAGs without the presence of a proteoglycan core protein. The administration of xylosides to living cells tends to result in a considerable amplification in GAG production, and the xylosides can, therefore, be used as analytical tools to study the GAG produced by a certain cell type. One of the most common ways to analyze the GAGs structurally is by disaccharide analysis, which involves depolymerization of the GAGs into disaccharides, fluorescent labeling of the disaccharides with 2-aminoacridone, and quantification using high-pressure liquid chromatography (HPLC). Here, we describe the procedure of producing xyloside-primed GAGs and how to study them structurally by disaccharide analysis. © 2022, Springer Science+Business Media, LLC, part of Springer Nature.
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| 343. |
- Persson, Helena, et al.
(author)
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Antibody validation by immunoprecipitation followed by mass spectrometry analysis
- 2017
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In: Methods in Molecular Biology. - New York, NY : Humana Press Inc.. ; 1575, s. 175-187
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Conference paper (peer-reviewed)abstract
- We describe a mass spectrometry-based approach for validation of antibody specificity. This method allows validation of antibodies or antibody fragments, against their endogenous targets. It can assess if the antibody is able to bind to its native antigen in cell lysates among thousands of other proteins, DNA, RNA, and other cellular components. In addition, it identifies other proteins the antibody is able to immunoprecipitate allowing for the assessment of antibody specificity and selectivity. This method is easily scalable, adaptable to different cell lines and conditions and has been shown to be reproducible between multiple laboratories.
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| 344. |
- Pettersson, Mats, et al.
(author)
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Capacitating epistasis--detection and role in the genetic architecture of complex traits.
- 2015
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In: Methods in Molecular Biology. - New York, NY : Springer New York. - 1064-3745 .- 1940-6029. ; 1253:1253, s. 185-196
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Journal article (peer-reviewed)abstract
- Here, we discuss the potential role of capacitating epistasis in the genetic architecture of complex traits. Two alternative methods for identifying such gene-gene interactions in genetic association studies-mapping of variance controlling loci and the variance plane ratio (VPR) method-are introduced. An overview of the theoretical foundation of the methods is presented together with a discussion on their implementation and available software for performing these analyses. We conclude by highlighting a few examples of capacitating epistasis described in the literature and its potential impacts on the genetics of complex traits.
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| 345. |
- Pfeiffer, Indriati, 1974, et al.
(author)
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Formation of pit-spanning phospholipid bilayers on nanostructured silicon dioxide surfaces for studying biological membrane events
- 2013
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In: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1940-6029 .- 1064-3745. - 9781627033350 ; 991, s. 113-125
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Book chapter (other academic/artistic)abstract
- Zwitterionic phospholipid vesicles are known to adsorb and ultimately rupture on flat silicon dioxide (SiO 2 ) surfaces to form supported lipid bilayers. Surface topography, however, alters the kinetics and mechanistic details of vesicles adsorption, which under certain conditions may be exploited to form a suspended bilayer. Here we describe the use of nanostructured SiO 2 surfaces prepared by the colloidal lithography technique to scrutinize the formation of suspended 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC) lipid bilayers from a solution of small unilamellar lipid vesicles (SUV s ). Atomic force microscopy (AFM) and quartz crystal microbalance with dissipation monitoring (QCM-D) were employed to characterize nanostructure fabrication and lipid bilayer assembly on the surface. © 2013 Springer Science+Business Media New York.
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| 346. |
- Pielberg, Gerli, et al.
(author)
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Gene copy number detection in animal studies
- 2007
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In: Methods in Molecular Biology. - New Jersey : Humana Press. - 1064-3745 .- 1940-6029. ; 373, s. 147-156
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Journal article (peer-reviewed)abstract
- Sensitive methods for the quantification of DNA fragments can be used to study an individual's genetic constitution for duplicated regions of the genome or to determine the relative proportion of different DNA fragments in heterogeneous samples such as pooled DNA from different individuals or in samples in which a fraction of the chromosomes carry a mutation. Here, we describe how we are using Pyrosequencing for this purpose. In Subheading 3., we describe a sensitive method that can be used to quantify the relative proportion of X- and Y-carrying sperm after sperm sorting in cattle. We also discuss our method for determining the copy numbers at a duplicated locus. This method has been applied to study genetic variation at the KIT locus in pigs, which have a major effect on coat color variation in this species.
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| 347. |
- Poças, Juliana, et al.
(author)
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Analysis of Extracellular Vesicle-Associated Proteoglycans
- 2023
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In: Methods in molecular biology (Clifton, N.J.). - New York, NY : Springer US. - 1940-6029. ; 2619, s. 125-139
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Journal article (peer-reviewed)abstract
- Extracellular vesicles (EVs) have emerged as a central mechanism of intercellular communication in physiology and disease. EVs participate in paracrine exchange of nucleic acids as well as lipids, proteins, and glycans to elicit a complex biological response in target cells. Proteoglycans (PGs) are widely expressed in EV-producing cells and are sorted to the membrane of secreted EVs to participate in some of the key processes in EV-mediated signaling. Most notably, PGs mainly of the heparan sulfate (HS) type are involved in EV biogenesis and cellular uptake of EVs through endocytic processes. EV-associated PGs may serve as short- and long-range chaperones of signaling molecules with potential implications for intercellular information exchange, most importantly in cancer development. This motivates the development of approaches targeting EV-HSPG interactions as a strategy in cancer treatment. Moreover, the importance of PG remodeling and alterations in their expression in cancer, together with the fact that EVs mimic their cell or tissue of origin, point at an important role of EV-associated PGs as disease biomarkers. Here, we provide methodological insights into the analysis of EV-PGs isolated from cell cultures as well as patient plasma liquid biopsy.
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| 348. |
- Pohl, Carsten, et al.
(author)
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Genome editing in penicillium chrysogenum using cas9 ribonucleoprotein particles
- 2018
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In: Methods in Molecular Biology. - New York, NY : Springer New York. - 1940-6029 .- 1064-3745. ; , s. 213-232
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Book chapter (other academic/artistic)abstract
- Several CRISPR/Cas9 tools have been recently established for precise genome editing in a wide range of filamentous fungi. This genome editing platform offers high flexibility in target selection and the possibility of introducing genetic deletions without the introduction of transgenic sequences. This chapter describes an approach for the transformation of Penicillium chrysogenum protoplasts with preassembled ribonucleoprotein particles (RNPs) consisting of purified Cas9 protein and in vitro transcribed single guide RNA (sgRNA) for the deletion of genome sequences or their replacement with alternative sequences. This method is potentially transferable to all fungal strains where protoplasts can be obtained from.
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| 349. |
- Polisetti, Naresh, et al.
(author)
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The artificial cornea
- 2013
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In: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1064-3745 .- 1940-6029. ; 1014, s. 45-52
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Journal article (peer-reviewed)abstract
- Human corneal transplantation to date suffers from the shortage of good-quality donor tissue, and in some conditions, allografting is contraindicated. A range of artificial replacements to donor allograft corneas have been developed. These range from keratoprostheses (KPro) that replace basic corneal functions of light transmission and protection to regenerative medicine strategies for regenerating one or more layers of the human cornea. This chapter reviews the advances made in developing artificial corneas or more accurately, artificial alternatives to donor allograft corneas for ocular application.
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| 350. |
- Pronk, Kees-Jan, et al.
(author)
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Flow cytometry-based identification of immature myeloerythroid development.
- 2011
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In: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1940-6029. ; 699, s. 275-293
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Journal article (peer-reviewed)abstract
- Precursor cells of the myeloerythroid cell lineages give rise to mature cells of the granulocyte, monocyte, erythroid, and/or thrombocytic lineages. High-resolution profiling of the developmental stages, from hematopoietic stem cells to mature progeny, is important to study and understand the underlying mechanisms that guide various cell fate decisions. In addition, this approach provides greater insights into pathogenic events such as leukemia, diseases that are most often characterized by halted differentiation at defined immature precursor levels. In this chapter, we provide protocols and discuss approaches concerning the analysis and purification of immature myeloerythroid lineages by multiparameter flow cytometry. Although recent data have demonstrated the feasibility of similar approaches also for the human system, we will focus our chapter on C57BL/6 mice, in which immunophenotypic applications have been most widely developed. This should also allow for its application in genetically modified models on this background. For maximal reproducibility, all protocols described have been established using reagents from commercial vendors to be analyzed on a three-laser flow cytometer with factory standard configuration.
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