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Competitive RT-PCR to Quantify Small Amounts of mRNA

Monitoring cytokine responses to various stimuli or determining the expression pattern of cytokine mRNAs requires sensitive technologies for cytokine mRNA quantitation where there is a limited quantity of material, for example, when working with biopsies. Owing to their amplif ...

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Purification, Identification, and Synthesis of Chemokines

Chemokines, i.e., low-molecular-weight chemotactic cytokines, are generally produced in very small amounts. Hence, liters of conditioned medium from primary cells or cell lines are necessary as start material to obtain microgram amounts of natural chemokine (see Note 1). A substanti ...

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In SituHybridization for Cytokines in Human Tissue Biopsies

In situ hybridization (ISH) involves hybridization of labeled nucleic acid probes with the target mRNA. As cytokines are soluble mediators exported from the cells in which they are produced, ISH is a powerful technique that can be used to identify the exact identity and location of the cells that p ...

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Receptor Isolation and Characterization: From Protein to Gene

Affinity chromatography as it is known today, namely, the concept and the immense power of biorecognition as a means of purification, was introduced in 1968 by Cuatracasas, Wilchek, and Anfinsen (1). This technique is used in 60% of all purification protocols (2). Almost any given biomolecule that ...

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Crystallization of Cytokine-Receptor Complexes

Crystallography is the most powerful method for determining the three-dimensional structure of complicated biological molecules. As the technology has improved, larger and more complicated structures involving previously unobtainable proteins have become available. ...

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Biosensor Analysis of Receptor-Ligand Interactions

Cytokines trigger growth factor responses through interaction with cell surface receptors. Determining the quantitative properties of cytokine-receptor interactions can help lead to mechanistic understanding of receptor activation as well as strategies to inhibit the i ...

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Site-Directed Mutagenesis to Determine Structure Function Relationships in Streptococcus pneumoniae Penicillin-Binding Protein Genes

βlactam resistance in clinical isolates of Streptococcus pneumoniae arises by only one route, the reduction of the affinity of the penicillin-binding proteins (PBPs) for βlactams. The pneumococcus possesses five high molecular weight PBPs (PBP1A, 1B, 2A, 2B, and 2X) which are involved in t ...

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Purification of DNA Topoisomerases and Inhibition by Fluoroquinolones

Fluoroquinolones act by inhibiting the essential type II topoisomerases, DNA gyrase and topoisomerase IV (1). DNA gyrase is a tetramer composed of 2 A subunits and 2 B-subunits encoded for by gyrA and gyrB, respectively. This enzyme is the only enzyme known that is capable of negatively supercoil ...

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Detection of Low Affinity Penicillin-Binding Protein Variants in Streptococcus pneumoniae

Penicillin-resistance in Streptococcus pneumoniae is mediated by altered penicillin-target enzymes, the penicillin-binding proteins or PBPs. PBPs interact withβ-lactam antibiotics by forming an active penicilloyl-PBP complex via an active site serine. This complex is en ...

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Mobilization of Transposons: Rationale and Techniques for Detection

The ability to share genetic information with other bacteria represents one of the most important adaptive mechanisms available to bacteria pathogenic for humans. The exchange of many different types of genetic information appears to occur frequently and exchange of determinants ...

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Preparation and Testing of Monoclonal Antibodies to Recombinant Proteins

Monoclonal antibodies (MAbs) are essential reagents for the isolation, identification, and cellular localization of specific gene products and for aiding in the determination of their macromolecular structure. They can also help in identifying the function of the protein. Altho ...

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A Simple Hollow-Fiber Bioreactor for the In-House Production of Monoclonal Antibodies

Monoclonal antibodies (MAbs) have traditionally been produced in quantity by transfer of the hybridomas to the peritoneal cavity of mice and subsequent recovery of MAb from the ascitic fluid. Legislation in many countries, however, now limits the use of animals for such purposes. Although ...

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Screening of Monoclonal Antibodies Using Antigens Labeled with Acetylcholinesterase

The production of large quantities of monoclonal antibodies (MAbs) of predetermined specificity has been rendered possible by the pioneering work of K�hler and Milstem (1) These workers have shown that lymphocytes can be immortalized and subsequently cultured after so1atic fusion ...

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Synthesis of Peptides for Use as Immunogens

An increasing problem in cell and molecular biology is the preparation of antibodies specific to proteins that are present in minute quantities within cells or tissues. With the advent of recombinant DNA technology, it is now often possible to deduce the primary amino acid sequence of a polypep ...

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Use of Proteins Blotted to Polyvinylidene Difluoride Membranes as Immunogens

The great analytical power of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) makes it one of the most effective tools of protein chemistry and molecular biology. In the past, there have been many attempts to convert the technique from analytical to preparative s ...

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Purification of Glycoproteins and Their Use as Immunogens

Glycosylation is a major posttranslational modification that produces heterogeneity in a protein and results in a group of structurally different species called glycoforms. Protein-associated oligosaccharides are large, structurally diverse molecules, and the biolog ...

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Detection and Clinical Relevance of Antibodies After Transplantation

Until recently, the role of antibodies in graft failure has been hampered by poor methods of defining specificity. Development of solid phase assays using purified major histocompatibility complex (MHC) molecules has greatly advanced our ability to monitor anti-human leukocyte an ...

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11 Reprogramming the Immune System Using Antibodies

Tolerance induction induced by monoclonal antibodies or co-receptor blockade is robust enough to resist breakdown by adoptive transfer of lymphocytes. Such resistance, the hallmark of dominant tolerance, is mediated by CD4 + regulatory T cells. CD4 + CD25 + T cells inhibit lymphopenia-m ...

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Proteomics and Laser Microdissection

Two-dimensional gel electrophoresis (2-DE) combined with protein identification by mass spectrometry (MS) is currently the method of choice in the majority of proteomic projects. Novel gel-free technologies have been developed but 2-DE remains the technique of choice for quantit ...

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In Vitro Assays for Immune Monitoring in Transplantation

Because immune responses to transplant allografts are the main drivers of rejection, the ability to accurately quantitate antidonor immunity is an important goal in clinical transplantation. These allow for the prediction of presensitization to the transplanted tissue and the id ...

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