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丁香实验推荐阅读
Hydrophobic Interaction Chromatography to Analyze Glycoproteins

Hydrophobic interaction chromatography (HIC) is one of many separation techniques that can be used to analyze proteins. The separation mechanism is based on the adsorption of the hydrophobic region of the protein to the hydrophobic ligands attached to the column in the presence of high salt. ...

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Characterization of Glycoprotein Biopharmaceutical Products by Caliper LC90 CE-SDS Gel Technology

Over the last decade, science has greatly improved in the area of protein sizing and characterization. Efficient high-throughput methods are now available to substitute for the traditional labor-intensive SDS-PAGE methods, which alternatively take days to analyze a very limited nu ...

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Application of Capillary Electrophoresis in Glycoprotein Analysis

Capillary electrophoresis (CE) is a versatile analytical method used to characterize glycoproteins. We have used several modes of CE separation such as CE-SDS gel, imaged capillary isoelectric focusing (icIEF), and capillary zone electrophoresis (CZE) to study therapeutic glyc ...

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Assessing Fc Glycan Heterogeneity of Therapeutic Recombinant Monoclonal Antibodies Using NP-HPLC

Recombinant monoclonal antibodies (rMAbs) are becoming major human therapeutics to treat life-threatening diseases such as cancer. These rMAbs are produced using either in vitro cell culture processes or transgenic technology in animals or plants. Glycans present in the Fc region c ...

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Engineering the Baculovirus Genome to Produce Galactosylated Antibodies in Lepidopteran Cells

Nowadays, recombinant proteins are used with great success for the treatment of a variety of medical conditions, such as cancer, autoimmune, and infectious diseases. Several expression systems have been developed to produce human proteins, but one of their most critical limitations is t ...

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N-Glycosylation Humanization for Production of Therapeutic Recombinant Glycoproteins in Saccharomyces cerevisiae

The production of therapeutic recombinant glycoproteins deals with three main issues: cost, production capacities, and glycosylation. Nowadays, such proteins are expressed in various complex expression systems (CHO, bacteria, etc.); the processes related to those production ...

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Glycoengineered Pichia-Based Expression of Monoclonal Antibodies

Currently, mammalian cells are the most commonly used hosts for the production of therapeutic monoclonal antibodies (mAbs). These hosts not only secrete mAbs with properly assembled two heavy and two light chains but also deliver mAbs with a glycosylation profile that is compatible with a ...

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Engineering a Human-Like Glycosylation to Produce Therapeutic Glycoproteins Based on 6-Linked Sialylation in CHO Cells

When recombinant glycoproteins for therapeutic use are to be produced on an industrial scale, there is a crucial need for technologies that can engineer fast-growing stable cells secreting the protein drug at a high rate and with a defined and safe glycosylation profile. Current cell lines ap ...

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Engineering of Therapeutic and Diagnostic O-Glycans on Recombinant Mucin-Type Immunoglobulin Fusion Proteins Expressed in CHO Cells

Metabolic engineering of mammalian cells for optimized glycosylation is usually done to improve activity and the pharmacokinetic features of glycoprotein therapeutics. The field is mainly focused around engineering of N-glycans. We have created a platform in which recombinant ...

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Combined MicroRNA In Situ Hybridization and Immunohistochemical Detection of Protein Markers

MicroRNAs are short (18-23 nucleotides) non-coding RNAs involved in posttranscriptional regulation of gene expression through their specific binding to the 3’UTR of mRNAs. MicroRNAs can be detected in tissues using specific locked nucleic acid (LNA)-enhanced probes. The charact ...

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Bright-Field In Situ Hybridization Methods to Discover Gene Amplifications and Rearrangements in Clinical Samples

Brightfield in situ hybridization (BISH) applications have significant advantages over traditional fluorescence in situ hybridization (FISH). BISH slides can be analyzed using a regular microscope while FISH slides require the use of a specialized fluorescence microscope. B ...

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In Vivo Target Validation by Inducible RNAi in Human Xenograft Mouse Models

Proper target selection and validation are crucial to the discovery of new anti-cancer agents. Since tumors depend on a suitable microenvironment for their growth, once a putative target has been identified, its validation should be performed whenever possible in vivo. This chapter dea ...

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Target Validation in Mice by Constitutive and Conditional RNAi

Gene silencing by RNA interference (RNAi) has become a standard method for the characterization of gene function in mammalian cells. Short hairpin (sh) RNAs expressed from stably integrated vectors mediate gene knockdown both in cultured cells and in mice, presenting a fast alternative to ...

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Genetically Engineered Animal Models for In Vivo Target Identification and Validation in Oncology

In vitro approaches using human cancer cell lines aimed to identify and validate oncology targets, have pinpointed a number of key targets and signalling pathways which control cell growth and cell death. However, tumors are more than insular masses of proliferating cancer cells. Instead t ...

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The Neurosphere Assay Applied to Neural Stem Cells and Cancer Stem Cells

The discovery of neural stem cells (NSCs) in the mammalian brain has raised many expectations as these unique cells might recapitulate different neurological diseases, including brain tumors, both from a functional and molecular perspective. Proper in vitro culturing of NSCs has emer ...

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Tumor Spheroid-Based Migration Assays for Evaluation of Therapeutic Agents

Cell migration is a key hallmark of malignant cells that contributes to the progression of cancers from a primary, localized mass to an invasive and/or metastatic phenotype. Traditional methods for the evaluation of tumor cell migration in vitro generally employ two-dimensional (2D), ho ...

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Two-Dimensional vs. Three-Dimensional In Vitro Tumor Migration and Invasion Assays

Motility and invasion are key hallmarks that distinguish benign from malignant tumors, enabling cells to cross tissue boundaries, disseminate in blood and lymph and establish metastases at distant sites. Similar properties are also utilized by activated endothelial cells during ...

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A Cell Culture System That Mimics Chronic Lymphocytic Leukemia Cells Microenvironment for Drug Screening and Characterization

Chronic Lymphocytic Leukaemia (CLL) is an incurable disease that warrants new therapeutic treatments. CLL cells accumulate in the peripheral blood, in the bone marrow and in secondary lymphoid organs. Unlike circulating CLL cells, CLL cells resident in these last two compartments dis ...

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Reverse Phase Protein Microarrays and Their Utility in Drug Development

The majority of human diseases, including cancer, are characterized by abnormal protein function. Proteins regulate virtually every cellular process and exhibit multiple kinds of post-translational modification that modulate expression levels and activation states, su ...

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Systematic Analysis of Complex Signal Transduction Pathways Using Protein Fragment Complementation Assays

The state of a signal transduction pathway can be assessed by monitoring a given point, or a signaling node, of interest within that pathway by high content analysis. The activity at these nodes may be correlated with the general effect on cell number and morphology at the same time. Here we describe a meth ...

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