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
* coaching of fabrics beneficial 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
An advantage of this procedure that has lead to its use in many laboratories is that over 98% of all multivalent nucleotides, namely, substrate [a-32p]ATP, [a-32p]ADP, [32p]AMP, as well as any [32P]Pi, are retained in the capped Eppendorf assay tubes in the ZnCO 3 precipitate. The waste radioactivity is thus highly confined, occupies little volume, and can be allowed to decay off and then be dealt with as normal solid waste. , 1"~8relies on sodium dodecyl sulfate to inactivate adenylyl cyclase and on unlabeled ATP and unlabeled cAMP to overwhelm adenylyl cyclase and cAMP phosphodiesterases with substrate and thereby effectively prevent the further formation or degradation of [32p]cAMP.
The waste radioactivity is thus highly confined, occupies little volume, and can be allowed to decay off and then be dealt with as normal solid waste. , 1"~8relies on sodium dodecyl sulfate to inactivate adenylyl cyclase and on unlabeled ATP and unlabeled cAMP to overwhelm adenylyl cyclase and cAMP phosphodiesterases with substrate and thereby effectively prevent the further formation or degradation of [32p]cAMP. Reagents. 5, and approximately 100,000 cpm/ml [3H]cAMP, to monitor recovery of [32p]cAMP.
Place the dish on the table on insulating paper and aspirate the medium. (Caution: Medium is radioactive! 5 ml of test substance (37°), dissolved in incubation medium. Prepare zero time controls at this step (see next paragraph). Incubate at 37 °* in a CO2 incubator, for a time appropriate for the test substance. Stop Reaction. 1 mM cAMP. Continue to next paragraph. 1 mM cAMP to one set of triplicate wells. Continue to next paragraph. 2 mM cAMP. Cells usually do not come off, but if the BSA/serum of the medium shows up as turbidity, leave for 30 min at 4° for nucleotide extraction.