| 1. |
- Adamczyk, Barbara, 1985, et al.
(författare)
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High-throughput analysis of the plasma N-glycome by UHPLC
- 2017
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Ingår i: High-Throughput Glycomics and Glycoproteomics, Eds: Gordan Lauc & Manfred Wuhrer. - New York : Springer. - 1064-3745. - 9781493964918 ; , s. 97-108
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- The understanding of glycosylation alterations in health and disease has evolved significantly and glycans are considered to be relevant biomarker candidates. High-throughput analytical technologies capable of generating high-quality, large-scale glycoprofiling data are in high demand. Here, we describe an automated sample preparation workflow and analysis of N-linked glycans from plasma samples using hydrophilic interaction liquid chromatography with fluorescence detection on an ultrahigh-performance liquid chromatography (UHPLC) instrument. Samples are prepared in 96-well plates and the workflow features rapid glycoprotein denaturation, enzymatic glycan release, glycan purification on solid-supported hydrazide, fluorescent labeling, and post-labeling cleanup with solid-phase extraction. The development of a novel approach for plasma N-glycan analysis and its implementation on a robotic platform significantly reduces the time required for sample preparation and minimizes technical variation. It is anticipated that the developed method will contribute to expanding high-throughput capabilities to analyze protein glycosylation. © Springer Science+Business Media New York 2017.
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| 2. |
- Lisacek, F., et al.
(författare)
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Databases and associated tools for glycomics and glycoproteomics
- 2017
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Ingår i: High-Throughput Glycomics and Glycoproteomics. Eds: Gordan Lauc & Manfred Wuhrer. - New York : Springer. - 1064-3745. - 9781493964932 - 9781493964918 ; , s. 235-264
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- The access to biodatabases for glycomics and glycoproteomics has proven to be essential for current glycobiological research. This chapter presents available databases that are devoted to different aspects of glycobioinformatics. This includes oligosaccharide sequence databases, experimental databases, 3D structure databases (of both glycans and glycorelated proteins) and association of glycans with tissue, disease, and proteins. Specific search protocols are also provided using tools associated with experimental databases for converting primary glycoanalytical data to glycan structural information. In particular, researchers using glycoanalysis methods by U/HPLC (GlycoBase), MS (GlycoWorkbench, UniCarb-DB, GlycoDigest), and NMR (CASPER) will benefit from this chapter. In addition we also include information on how to utilize glycan structural information to query databases that associate glycans with proteins (UniCarbKB) and with interactions with pathogens (Sugar Bind). © Springer Science+Business Media New York 2017.
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| 3. |
- Ahlenius, Henrik
(författare)
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Preface
- 2021
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Ingår i: Methods in Molecular Biology. - 1064-3745. ; 2352
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 4. |
- Ahmad, Faiyaz, et al.
(författare)
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Adenovirus-mediated overexpression of murine cyclic nucleotide phosphodiesterase 3B
- 2005
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Ingår i: Methods in molecular biology (Clifton, N.J.). - New Jersey : Humana Press. - 1940-6029 .- 1064-3745. ; 307, s. 93-107
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- To construct the recombinant adenovirus vector containing the cDNA for recombinant mouse cyclic nucleotide phosphodiesterase 3B (mPDE3B), the cDNA for mPDE3B was subcloned into pACCMV.pLpA. Subsequently, this recombinant plasmid, pACCMV.mPDE3B, was cotransfected with pJM17 plasmid containing the adenoviral genome into 293 human embryonic kidney cells, and the replication-deficient adenovirus AdCMV.mPDE3B was generated via homologous recombination. Large-scale preparation of adenovirus yielded 10(11)-10(13) viral particles/mL and could be quantitated by real-time polymerase chain reaction using iCycler (Bio-Rad). Efficiency of gene transfer was assessed by infecting FDCP2 or H4IIE cells with a recombinant adenovirus expressing beta-galactosidase (beta-gal); greater than 75% of cells were infected. Expression of mPDE3B in H4IIE hepatoma cells, FDCP2 hematopoietic cells, and beta-cells from isolated pancreatic islets was detected by Western blot analysis. In lysates from FDCP2 cells and H4IIE hepatoma cells infected with recombinant adenoviral mPDE3B constructs, mPDE3B activity was increased 10- to 30-fold compared with the activity in lysates from cells infected with beta-gal adenovirus. Stimulation of FDCP2 cells infected with mPDE3B adenovirus with insulin (100 nM, 10 min) resulted in an approx 1.7-fold increase in endogenous PDE3B and recombinant wild-type PDE3B activities. Infection of rat pancreatic islets resulted in a 5- to 10-fold increase in PDE3B expression and activity and subsequent blunting of insulin secretion. Thus, adenovirus-mediated gene transfer is effective for studying expression and regulation of recombinant PDE3 in insulin-responsive cells as well as insulin-secreting cells.
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| 5. |
- Ahmed, RK, et al.
(författare)
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T-cell epitope mapping
- 2009
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Ingår i: Methods in molecular biology (Clifton, N.J.). - Totowa, NJ : Humana Press. - 1064-3745. ; 524, s. 427-38
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Tidskriftsartikel (refereegranskat)
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| 6. |
- Aits, Sonja, et al.
(författare)
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Methods to Detect Loss of Lysosomal Membrane Integrity
- 2019
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Ingår i: Autophagy : Methods and Protocols - Methods and Protocols. - New York, NY : Springer New York. - 1064-3745. - 9781493988730 ; 1880, s. 315-329
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Bokkapitel (refereegranskat)abstract
- Loss of lysosomal membrane integrity, often referred to as lysosomal membrane permeabilization (LMP), occurs in many instances of cell death either as an initiating or as an amplifying event. Currently, the best method for detecting LMP is the galectin puncta formation assay which can be used for a broad range of sample types, both fixed and live, is easy to perform, and highly sensitive. This method, which is similar to the widely used LC3 puncta formation assay for autophagy, is based on the translocation of galectins to damaged lysosomes resulting in a change from uniform to punctate staining pattern. Here, we provide protocols for the galectin puncta formation assay in fixed and live cells and for an alternative assay based on fluorescent dextran release from damaged lysosomes, which can be performed in parallel
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| 7. |
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| 8. |
- Allen, Marie, et al.
(författare)
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Mitochondrial D-loop and coding sequence analysis using pyrosequencing
- 2005
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Ingår i: Methods in Molecular Biology. - New Jersey : Humana Press. - 1064-3745 .- 1940-6029. ; 297, s. 179-196
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Tidskriftsartikel (refereegranskat)abstract
- In forensic casework analysis, mitochondrial deoxyribonucleic acid (DNA) often is used when the evidence material contains scarce amounts of DNA. Here, a mitochondrial DNA typing system for D-loop and coding region analysis based on pyrosequencing is described. Pyrosequencing is a real-time, single-tube sequencing-by-synthesis method, in which a cascade of enzymatic reactions yields detectable light. This pyrosequencing system has a higher resolution than the D-loop analysis performed routinely today as it also covers informative positions in the mitochondrial coding region. The system is composed of 16 polymerase chain reaction (PCR) fragments and 24 pyrosequencing reactions with a turn around time for a 96-well plate of less than 3 h after PCR.
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| 9. |
- Allen, Marie, et al.
(författare)
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Universal tag arrays in forensic SNP analysis.
- 2005
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Ingår i: Methods in Molecular Biology. - 1064-3745 .- 1940-6029. ; 297, s. 141-154
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Tidskriftsartikel (refereegranskat)abstract
- Microarray-based single nucleotide polymorphism (SNP) genotyping enables simultaneous and rapid detection of a large number of markers and is thus an attractive method for forensic individual acid identification. This assay relies on a one-color detection system and minisequencing in solution before hybridization to universal tag arrays. The minisequencing reaction is based on incorporation of a fluorescent dideoxynucleotide to a primer containing a tag-sequence flanking the position to be interrogated. This one-color system detects C and T polymorphisms in separate reactions on multiple polymerase chain reaction targets with the fluorophore TAMRA coupled to the respective dideoxynucleotide. After incorporation, tagged primer sequences are hybridized through their complementary sequence on the array, and positive signals are detected by a confocal laser-scanner.
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| 10. |
- Altai, Mohamed, et al.
(författare)
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Preparation of Conjugates for Affibody-Based PNA-Mediated Pretargeting.
- 2020
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer US. - 1064-3745 .- 1940-6029. ; 2105, s. 283-304, s. 283-304
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Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules are small engineered scaffold proteins suitable for in vivo tumor targeting. Radionuclide molecular imaging using directly radiolabelled affibody molecules provides excellent imaging. However, affibody molecules have a high renal reabsorption, which complicates their use for radionuclide therapy. The high renal reabsorption is a common problem for the use of engineered scaffold proteins for radionuclide therapy. Affibody-based PNA-mediated pretargeting reduces dramatically the absorbed dose to the kidneys and makes affibody-based radionuclide therapy possible. This methodology might, hopefully, solve the problem of high renal reabsorption for radionuclide therapy mediated by other engineered scaffold proteins.
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| 11. |
- Alvarez-Castro, Jose, et al.
(författare)
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Estimation and interpretation of genetic effects with epistasis using the NOIA model.
- 2012
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1064-3745 .- 1940-6029. ; 871, s. 191-204
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Tidskriftsartikel (refereegranskat)abstract
- We introduce this communication with a brief outline of the historical landmarks in genetic modeling, especially concerning epistasis. Then, we present methods for the use of genetic modeling in QTL analyses. In particular, we summarize the essential expressions of the natural and orthogonal interactions (NOIA) model of genetic effects. Our motivation for reviewing that theory here is twofold. First, this review presents a digest of the expressions for the application of the NOIA model, which are often mixed with intermediate and additional formulae in the original articles. Second, we make the required theory handy for the reader to relate the genetic concepts to the particular mathematical expressions underlying them. We illustrate those relations by providing graphical interpretations and a diagram summarizing the key features for applying genetic modeling with epistasis in comprehensive QTL analyses. Finally, we briefly review some examples of the application of NOIA to real data and the way it improves the interpretability of the results.
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| 12. |
- Andersson, Jan O.
(författare)
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Horizontal gene transfer between microbial eukaryotes.
- 2009
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Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1064-3745 .- 1940-6029. ; 532:4, s. 473-487
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Tidskriftsartikel (refereegranskat)abstract
- Comparative genomics have identified two loosely defined classes of genes: widely distributed core genes that encode proteins for central functions in the cell and accessory genes that are patchily distributed across lineages and encode taxa-specific functions. Studies of microbial eukaryotes show that both categories undergo horizontal gene transfer (HGT) from prokaryotes, but also between eukaryotic organisms. Intra-domain gene transfers of most core genes seem to be relatively infrequent and therefore comparatively easy to detect using phylogenetic methods. In contrast, phylogenies of accessory genes often have complex topologies with little or no resemblance of organismal relationships typically with eukaryotes and prokaryotes intermingled, making detailed evolutionary histories difficult to interpret. Nevertheless, this suggests significant rates of gene transfer between and among the three domains of life for many of these genes, affecting a considerably diversity of eukaryotic microbes, although the current depth of taxonomic sampling usually is insufficient to pin down individual transfer events. The occurrence of intra-domain transfer among microbial eukaryotes has important implications for studies of organismal phylogeny as well as eukaryote genome evolution in general.
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| 13. |
- André, Ingemar
(författare)
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Modeling the structure of helical assemblies with experimental constraints in Rosetta
- 2018
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer New York. - 1064-3745. ; 1764, s. 475-489
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Bokkapitel (refereegranskat)abstract
- Determining high-resolution structures of proteins with helical symmetry can be challenging due to limitations in experimental data. In such instances, structure-based protein simulations driven by experimental data can provide a valuable approach for building models of helical assemblies. This chapter describes how the Rosetta macromolecular package can be used to model homomeric protein assemblies with helical symmetry in a range of modeling scenarios including energy refinement, symmetrical docking, comparative modeling, and de novo structure prediction. Data-guided structure modeling of helical assemblies with experimental information from electron density, X-ray fiber diffraction, solid-state NMR, and chemical cross-linking mass spectrometry is also described.
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| 14. |
- Andreasson, Erik, et al.
(författare)
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Isolation of Apoplast
- 2017
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer New York. - 1064-3745 .- 1940-6029. ; 1511, s. 233-240
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Tidskriftsartikel (refereegranskat)abstract
- The apoplast can be described as the soluble fraction of the extracellular space of plant tissue, and it plays an important role in signaling, nutrient transport, and plant-pathogen interactions. In this protocol, we describe a method where leaves are infiltrated with phosphate buffer under vacuum. The apoplast can then be extracted by centrifugation and simultaneously collected in a protease inhibitor solution. Using this protocol, typically 3 mu g of apoplastic proteins can be obtained in a volume of 300 mu L from five potato leaflets, with minimal contamination by non-apoplastic proteins.
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| 15. |
- Archer, Amena, et al.
(författare)
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Expression Profiles of Estrogen-Regulated MicroRNAs in Cancer Cells.
- 2022
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer Nature. - 1064-3745 .- 1940-6029. ; 2418, s. 313-343
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Tidskriftsartikel (refereegranskat)abstract
- MicroRNAs play critical roles through their impact on posttranscriptional gene regulation. In cancer, they can act as oncogenes or tumor suppressors and can also function as biomarkers. Here, we describe a method for robust characterization of estrogen-regulated microRNA profiles. The activity of estrogen is mediated by two nuclear receptors, estrogen receptor alpha and estrogen receptor beta, and a transmembrane G-protein coupled estrogen receptor 1. This chapter details how to prepare cells for optimal estrogen response, directions for estrogen treatment, RNA extraction, different microRNA profiling approaches, and subsequent confirmations.
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| 16. |
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| 17. |
- Aslıyüce, Sevgi, et al.
(författare)
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Preparation of Staphylococcal Protein A Imprinted Supermacroporous Cryogel Beads
- 2022
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer US. - 1064-3745 .- 1940-6029. ; 2466, s. 261-273
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Bokkapitel (refereegranskat)abstract
- Protein A is the most commonly used ligand in IgG purification due to its specific binding to the Fc receptor of most immunoglobulins, making it commercially important. Molecular imprinting is a method based on the selective recognition of various molecules. Molecular imprinted polymers are materials that are easy to prepare, durable, cheap and have molecular recognition capability. Cryogels are prepared by radical polymerization in a partially frozen environment. The unique structure of cryogels combined with osmotic, chemical and mechanical stability make them attractive chromatography matrices for a variety of biological compounds/specimens (plasmids, pathogens, cells). In this protocol, protein A imprinted supermacroporous poly(2-hydroxyethyl methacrylate) cryogels were prepared in spherical form for protein A purification. The characterization of the prepared cryogels were made by swelling test, scanning electron microscopy (SEM), Fourier transform infrared spectrophotometer (FTIR), and Brunauer–Emmett–Teller (BET) surface area analysis. After characterization, optimum conditions for protein A adsorption were determined in the batch system. The maximum protein A adsorption capacity was determined after optimization of the imprinted cryogels. Protein A relative selectivity coefficients of imprinted cryogels were examined for both Fc and protein G. Protein A was isolated from the bacterial cell wall using fast performance liquid chromatography (FPLC). The separated protein A was determined by sodium dodecyl sulfate gel electrophoresis (SDS-PAGE). In the last stage, the reusability of the cryogel was examined.
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| 18. |
- Asp, Julia, 1973, et al.
(författare)
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Nonradioactive in situ hybridization on frozen sections and whole mounts.
- 2006
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Ingår i: Methods in molecular biology (Clifton, N.J.). - 1064-3745. ; 326, s. 89-102
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Tidskriftsartikel (refereegranskat)abstract
- Nonradioactive in situ hybridization offers a unique opportunity to study gene expression on samples with preserved histological information. This method makes it possible to locate not only where in a tissue a particular gene is expressed, but in many cases also in which specific cell type it is active. Here, we describe our current protocols for in situ hybridization on frozen sections or whole mounts of mouse embryos. The protocols included describe synthesis of a digoxigenin-labeled probe, tissue handling, hybridization of the probe to the mRNA expressed in the sample and signal detection.
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| 19. |
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| 20. |
- Aufschnaiter, Andreas, Dr. rer. nat. 1988-, et al.
(författare)
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Yeast Mitoribosome Purification and Analyses by Sucrose Density Centrifugation and Immunoprecipitation
- 2023
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Ingår i: Methods in Molecular Biology. - : Humana Press. - 1064-3745 .- 1940-6029. ; , s. 119-132, s. 119-132
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Mitochondrial protein biosynthesis is maintained by an interplay between the mitochondrial ribosome (mitoribosome) and a large set of protein interaction partners. This interactome regulates a diverse set of functions, including mitochondrial gene expression, translation, protein quality control, and respiratory chain assembly. Hence, robust methods to biochemically and structurally analyze this molecular machinery are required to understand the sophisticated regulation of mitochondrial protein biosynthesis. In this chapter, we present detailed protocols for immunoprecipitation, sucrose cushions, and linear sucrose gradients to purify and analyze mitoribosomes and their interaction partners.
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| 21. |
- Babrak, Lmar, et al.
(författare)
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Adaptive Immune Receptor Repertoire (AIRR) Community Guide to TR and IG Gene Annotation
- 2022
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Ingår i: Immunogenetics : Methods and Protocols - Methods and Protocols. - New York, NY : Springer US. - 1064-3745. - 9781071621158 - 9781071621141 ; 2453, s. 279-296
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Bokkapitel (refereegranskat)abstract
- High-throughput sequencing of adaptive immune receptor repertoires (AIRR, i.e., IG and TR) has revolutionized the ability to carry out large-scale experiments to study the adaptive immune response. Since the method was first introduced in 2009, AIRR sequencing (AIRR-Seq) has been applied to survey the immune state of individuals, identify antigen-specific or immune-state-associated signatures of immune responses, study the development of the antibody immune response, and guide the development of vaccines and antibody therapies. Recent advancements in the technology include sequencing at the single-cell level and in parallel with gene expression, which allows the introduction of multi-omics approaches to understand in detail the adaptive immune response. Analyzing AIRR-seq data can prove challenging even with high-quality sequencing, in part due to the many steps involved and the need to parameterize each step. In this chapter, we outline key factors to consider when preprocessing raw AIRR-Seq data and annotating the genetic origins of the rearranged receptors. We also highlight a number of common difficulties with common AIRR-seq data processing and provide strategies to address them.
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| 22. |
- Benetó, Noelia, et al.
(författare)
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Genome Editing Using Cas9-gRNA Ribonucleoprotein in Human Pluripotent Stem Cells for Disease Modeling
- 2022
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer US. - 1064-3745 .- 1940-6029. ; 2549, s. 409-425
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Bokkapitel (refereegranskat)abstract
- The discovery that the CRISPR/Cas9 system could be used for genome editing purposes represented a major breakthrough in the field. This advancement has notably facilitated the introduction or correction of disease-specific mutations in healthy or disease stem cell lines respectively; therefore, easing disease modeling studies in combination with differentiation protocols. For many years, variability in the genetic background of different stem cell lines has been a major burden to specifically identify phenotypes arising uniquely from the presence of the mutation and not from differences in other genomic regions. Here, we provide a complete protocol to introduce random indels in human wild type pluripotent stem cells using CRISPR/Cas9 in order to generate clonal lines with potential pathogenic alterations in any gene of interest. In this protocol, we use transfection of a ribonucleoprotein complex to diminish the risk of off-target effects, and select clonal lines with promising indels to obtain disease induced pluripotent stem cell lines.
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| 23. |
- Bhushan, Shashi, et al.
(författare)
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In vitro and in vivo methods to study protein import into plant mitochondria.
- 2007
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Ingår i: Methods Mol Biol. - 1064-3745. ; 390, s. 131-50
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Tidskriftsartikel (refereegranskat)abstract
- Plant mitochondria contain about 1000 proteins, 90-99% of which in different plant species are nuclear encoded, synthesized on cytosolic polyribosomes, and imported into the organelle. Most of the nuclear-encoded proteins are synthesized as precursors containing an N-terminal extension called a presequence or targeting peptide that directs the protein to the mitochondria. Here we describe in vitro and in vivo methods to study mitochondrial protein import in plants. In vitro synthesized precursor proteins can be imported in vitro into isolated mitochondria (single organelle import). However, missorting of chloroplast precursors in vitro into isolated mitochondria has been observed. A novel dual import system for simultaneous import of proteins into isolated mitochondria and chloroplasts followed by reisolation of the organelles is superior over the single import system as it abolishes the mistargeting. Precursor proteins can also be imported into the mitochondria in vivo using an intact cellular system. In vivo approaches include import of transiently expressed fusion constructs containing a presequence or a full-length precursor protein fused to a reporter gene, most commonly the green fluorescence protein (GFP) in protoplasts or in an Agrobacterium-mediated system in intact tobacco leaves.
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| 24. |
- Birgersson, Madeleine, et al.
(författare)
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Antibody Validation for Estrogen Receptor Beta
- 2022
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer Nature. - 1064-3745 .- 1940-6029. ; 2418, s. 1-23, s. 1-23
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies can cross-react with proteins other than their intended targets, and antibody-based applications can, if not properly validated, lead to flawed interpretations. When evaluating 13 anti-estrogen receptor beta (ERβ) antibodies in 2017, we concluded that only one of them was specific. Applying this antibody in immunohistochemistry of over 44 different normal human tissues and 20 types of cancers revealed ERβ expression in only a few selected tissues. This aligned with mRNA evidence but contradicted a large set of published literature. ERβ protein expression continues to be reported in tissues without clear support by mRNA expression. In this chapter, we describe how ERβ antibodies can be thoroughly validated and discuss selection of well-characterized positive and negative controls. The validation scheme presented is applicable for immunohistochemistry and Western blotting. The protocol includes evaluation of mRNA evidence, use of public databases, assessment of on- and off-target binding, and an optional step for corroboration with immunoprecipitation and mass spectrometry.
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| 25. |
- Bojar, Daniel
(författare)
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Construction of Caffeine-Inducible Gene Switches in Mammalian Cells
- 2021
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Ingår i: Mammalian Cell Engineering. - New York, NY : Springer. - 1064-3745. ; , s. 159-168
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Controlling gene expression in mammalian cells constitutes one of the core principles of mammalian synthetic biology. Especially for cell-based therapies, inducers of gene expression which demonstrate the highest degree of safety and patient adherence are needed. In this chapter, I describe methods to implement caffeine-controlled gene expression systems into mammalian cells. Using an array of different implementation strategies, from reconstituting transcription factors to activating endogenous signaling pathways, allows for a wide range of sensitivity and capacity of the resulting caffeine-responsive gene switches. © 2021, Springer Science+Business Media, LLC, part of Springer Nature.
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