| 1. |
- 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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| 2. |
- 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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| 3. |
- 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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| 4. |
- 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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| 5. |
- 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. - 1940-6029 .- 1064-3745. ; 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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| 6. |
- 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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| 7. |
- Canals, Isaac, et al.
(författare)
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CRISPR/Cas9 Genome Engineering in Human Pluripotent Stem Cells for Modeling of Neurological Disorders
- 2021
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Ingår i: Neural Reprogramming : Methods and Protocols - Methods and Protocols. - New York, NY : Springer US. - 1940-6029 .- 1064-3745. - 9781071616017 - 9781071616000 ; 2352, s. 237-251
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Bokkapitel (refereegranskat)abstract
- Recent advances in genome editing have brought new hopes for personalized and precision medicine but have also dramatically facilitated disease modeling studies. Combined with reprogramming approaches, stem cells and differentiation toward neural lineages, genome engineering holds great potential for regenerative approaches and to model neurological disorders. The use of patient-specific induced pluripotent stem cells combined with neural differentiation allows studying the effect of specific mutations in different brain cells. New genome editing tools such as CRISPR/Cas9 represent a step further by facilitating the introduction or correction of specific mutations within the same cell line, thus eliminating variability due to differences in the genetic background. Here, we present a step-by-step protocol from design to generation of human pluripotent stem cell lines with specific mutations introduced or corrected with CRISPR/Cas9 gene editing that can be used in combination with transcription factor-based protocols to dissect underlying mechanisms of neurological disorders.
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| 8. |
- Chapellier, Marion, et al.
(författare)
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Arrayed molecular barcoding of leukemic stem cells
- 2021
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Ingår i: Leukemia Stem Cells. - New York, NY : Springer US. - 1940-6029 .- 1064-3745. ; 2185, s. 345-359
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Bokkapitel (refereegranskat)abstract
- Functional screens on cancer cells using compound or protein libraries are usually performed in vitro. However, to assess the effects on leukemia stem cells (LSCs) in a screening setting, methodologies that allow for a high-throughput in vivo readout of leukemia-initiating activity are needed. One experimental approach to solve this issue is to genetically label, also referred to as “barcoding,” the leukemia cells in an arrayed format prior to exposing them to separate experimental conditions. The cells can then be pooled and injected into mice for competitive readout of leukemia-initiating activity. Here, we describe a procedure for combining lentiviral arrayed molecular barcoding of leukemia cells with next-generation sequencing, to enable screens on leukemia cells ex vivo followed by an in vivo competitive readout of LSC function. This methodology can also be applied to other model systems in which a competitive in vivo readout of cells is needed.
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| 9. |
- Dam, Tommy, et al.
(författare)
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Fluorescence-Based Measurements of Two-Dimensional Affinity in Membrane Interfaces
- 2023
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Ingår i: Methods in Molecular Biology. - 1064-3745 .- 1940-6029. ; 2654, s. 25-40
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Bokkapitel (refereegranskat)abstract
- Binding between ligands and receptors across cell contacts influences a range of biological processes including the formation of the immune synapse. The dissociation constant (Kd = 1/affinity) of the interaction corresponds to the concentration of ligands where half of the receptors in the contact have bound a ligand. In this chapter, we outline how to measure this two-dimensional affinity using model cell membranes called supported lipid bilayers (SLBs) functionalized with fluorescently labeled ligands that bind to cells containing the corresponding receptor. The affinity is calculated from the accumulation of ligands at the cell-SLB interface, while the use of different fluorescent tags, and/or unlabeled molecules, makes it possible to include various binding pairs in the contact to better mimic the conditions of binding in vivo.
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| 10. |
- Dupard, Steven J., et al.
(författare)
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3D Engineering of Human Hematopoietic Niches in Perfusion Bioreactor
- 2021
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Ingår i: Methods in Molecular Biology. - New York, NY : Springer US. - 1940-6029 .- 1064-3745. ; 2308, s. 253-262
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Bokkapitel (refereegranskat)abstract
- The hematopoietic microenvironment, also referred to as hematopoietic niche, is a functional three-dimensional (3D) unit of the bone marrow (BM) that planar culture systems cannot recapitulate. Existing limitations of 2D protocols are driving the development of advanced 3D methodologies, capable of superior modeling of the native organization and interactions between hematopoietic cells and their niche. Hereafter we describe the use of a 3D perfusion bioreactor for in vitro generation of human hematopoietic niches. The approach enables the recapitulation of the interactions between hematopoietic stem and progenitor cells (HSPCs), mesenchymal cells (MSCs), and their extracellular matrix in a 3D relevant setting. This was shown to support the functional maintenance of blood populations, self-distributing in the system compartments depending on their differentiation status. Such 3D niche modeling represents an advanced tool toward uncovering human hematopoiesis in relation to its host microenvironment, for both fundamental hematopoiesis and personalized medicine applications.
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