By John N. Abelson, Melvin I. Simon, Roger A. Johnson, Jackie D. Corbin
This quantity emphasizes equipment for the assay, purification, and characterization of adenylyl cyclases, guanine nucleotide-dependent regulatory proteins (G proteins), and guanylyl cyclases.
* Adenylyl cyclase: decision of activity
* training of fabrics worthy 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
* coaching of fabrics used for learn of the cyclase
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Additional resources for Adenylyl Cyclase, G Proteins, and Guanylyl Cyclase
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. An option for combining Stages I and 2 is also listed in this section. This option is particularly useful when only a3ZP-labeled nucleotides or cyclic nucleotides are to be synthesized. Stage 1 Protocol. The volume of the [32P]Pi (the volume of 10 mCi of [32p]pi is usually about 50/zl) dictates the volume in which Stage 1 and other stages are carried out.
CAMP is eluted from the alumina columns directly into scintillation vials. The volume of elution buffer used depends on whether unlabeled cAMP or [3H]cAMP is used to quantitate sample recovery and on the types of vials used in the scintillation counter. It is important to use sufficient buffer to elute all the cAMP as well as to optimize counter efficiency, which is dictated by the geometry of the counter phototubes. For example, if recovery is monitored with unlabeled cAMP and [32p]cAMP is determined by Cerenkov radiation in 20 ml counting vials, [32p]cAMP is eluted from alumina columns with 8 ml of 100 mM Tris-C1.
5 × Stage 1 reaction mixture (see Table I) and enzyme-DTT mixture (see Table II) are prepared from the reagent stocks or enzyme mix just prior to use. The final concentrations of reagents and enzymes in Stage 1 have been chosen such that complete conversion of [32P]P i to [y-32p]ATP occurs with minimal contamination of [32p]p i with unlabeled Pi. 2 M ammonium sulfate. b The enzymes are mixed in the proportions shown and stored at 4 °. This stock enzyme mixture is stable for up to 1 year. e The enzyme-DTT mixture is prepared just before use by adding 5/zl of ammonium sulfate-depleted stock enzyme mix to 65/zl of a buffer containing 50 m M Tris-HCl, pH 8, and 5 m M DTT.
Adenylyl Cyclase, G Proteins, and Guanylyl Cyclase by John N. Abelson, Melvin I. Simon, Roger A. Johnson, Jackie D. Corbin