Production of reagents and optimization of methods for studying calmodulin-binding proteins
| Autor: | Thierry Soldati, Bettina Ulbricht |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 1999 |
| Předmět: |
Cytosol/metabolism
Calmodulin-Binding Proteins/metabolism Cloning Molecular/methods animal structures Calmodulin Immunoprecipitation Blotting Western Fluorescent Antibody Technique Recombinant Proteins/biosynthesis/isolation & purification/metabolism Biology Dictyostelium discoideum Chromatography Affinity law.invention Cytosol Affinity chromatography law Dictyostelium/genetics/metabolism Escherichia coli Animals Dictyostelium Cloning Molecular Polyacrylamide gel electrophoresis biology.organism_classification Chromatography Ion Exchange Calmodulin-binding proteins Recombinant Proteins Biochemistry Calmodulin/genetics/isolation & purification/metabolism Polyclonal antibodies biology.protein Recombinant DNA Calmodulin-Binding Proteins Electrophoresis Polyacrylamide Gel Indicators and Reagents Biotechnology |
| Zdroj: | Protein Expression and Purification, Vol. 15, No 1 (1999) pp. 24-33 Protein Expression and Purification |
| ISSN: | 1046-5928 |
| Popis: | Owing to subtle but potentially crucial structural and functional differences between calmodulin (CaM) of different species, the biochemical study of low-affinity CaM-binding proteins from Dictyostelium discoideum likely necessitates the use of CaM from the same organism. In addition, most of the methods used for identification and purification of CaM-binding proteins require native CaM in nonlimiting biochemical quantities. The gene encoding D. discoideum CaM has previously been cloned allowing production of recombinant protein. The present study describes the expression of D. discoideum CaM in Escherichia coli and its straightforward and rapid purification. Furthermore, we describe the optimization of a complete palette of assays to detect as little as nanogram quantities of proteins binding CaM with middle to low affinities. Purified CaM was used to raise high-affinity polyclonal antibodies suitable for immunoblotting, immunofluorescence, and immunoprecipitation experiments. The purified CaM was also used to optimize a specific and sensitive nonradioactive CaM overlay assay as well as to produce a high-capacity CaM affinity chromatography matrix. The effectiveness of this methods is illustrated by the detection of potentially novel D. discoideum CaM-binding proteins and the preparatory purification of one of these proteins, a short tail myosin I. |
| Databáze: | OpenAIRE |
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