By John N. Abelson, Melvin I. Simon, Roger A. Johnson, Jackie D. Corbin
This quantity emphasizes tools for the assay, purification, and characterization of adenylyl cyclases, guanine nucleotide-dependent regulatory proteins (G proteins), and guanylyl cyclases.
* Adenylyl cyclase: decision of activity
* education of fabrics necessary in purification of parts of hormonally responsive cyclase systems
* Purification guanine nucleotide-dependent regulatory proteins
* Purification and characterization of g proteins: labeling and quantitating of G proteins
* Reconstitution guanylyl cyclase: assay
* practise of fabrics used for research of the cyclase
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Extra resources for Adenylyl Cyclase, G Proteins, and Guanylyl Cyclase
With a plastic scoop or a large disposable plastic syringe from which the alumina is allowed to drain). Elution buffers: The original method of Salomon et al. 5. 5. The purpose of the buffer is to elute cyclic nucleotides. Since eluate from the Dowex 50 columns is acidic, which enhances adsorption of cyclic nucleotides to alumina, elution of cyclic nucleotides is achieved principally through an increase in the pH of the buffer rather than through increased ionic strength. Consequently, there is probably wide latitude in buffer choice here.
Some of these considerations are dealt with below. Stopping with Zinc Acetate/Na2CO3/cAMP or/[3H]cAMP The use of coprecipitation or adsorption of nucleotides with inorganic salts dates from an early assay for adenylyl cyclase developed by Krishna et al. ,24who used a combination of column chromatography on Dowex 50 and precipitation with ZnSO4 and Ba(OH)2, yielding the insoluble salts of BaSO 4 and Zn(OH)E, which adsorb phosphomonoesters and polyphosphates but not cyclic nucleotides. A disadvantage in the use of ZnSO4/ Ba(OH)2 is that cAMP may be formed nonenzymatically from ATP at alkaline pH, especially at elevated temperatures, leading to variable and high blank values.
Carrier-free [32p]pi should be ordered with instructions to the supplier that the material be shipped in the smallest possible volume (without dilution), ff the [32p]p i arrives in a greater volume (1 ml is standard) the procedure can still be carried out by scaling up the volumes of reagents and enzymes listed below. We use [32P]Pi in 20 mM HC1 from New England Nuclear (Boston, MA) routinely. 5 × Stage 1 reaction mixture are listed in Table I. The enzymes necessary are listed in Table II. The reagents should be prepared just prior to initiating Stage 1.