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
* guidance of fabrics priceless in purification of elements 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
* education of fabrics used for learn of the cyclase
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Additional resources for Adenylyl Cyclase, G Proteins, and Guanylyl Cyclase
Apparatus for column chromatography. of the racks supporting the alumina columns should allow the eluate from them to drip directly into scintillation vials. Carbon filters for the removal of adsorbable radioactive materials from column eluates are Gelman Ann Arbor, MI (#12011) Carbon Capsules, each containing 100 g activated charcoal. Procedure. Two alternative procedures are described, the choice of which depends on the quality of 32p-labeled substrate. The first procedure should be adequate with all but the poorest quality substrate and the second procedure should lower blank values further if necessary.
6 x 4 cm). After each use, Dowex 50 columns are regenerated by washing with 5 ml of 1 N HC1, then stored until reused. Before use the columns are washed 3 times with 10 ml of water. Columns can be reused dozens of times. If flow rates decrease, columns should be regenerated with NaOH, water, and HCI as above. , Bio-Rad AG7, 100-200 mesh; Sigma WN-3; ICN (Costa Mesa, CA) Alumina N, Super I]: The source of A1203 is less critical with the two-column procedure than if it is used alone. , with a plastic scoop or a large disposable plastic syringe from which the alumina is allowed to drain).
At this stage, prelabeled cells are stimulated to form [3H]cAMP. 25 ml. Prepare zero time controls at this step (see next paragraph). Incubate at 37°* for a time appropriate for the test substance. Stop Reaction. 1 mM cAMP to extract labeled nucleotides from the cell pellet. Resuspend the cell pellets for efficient extraction of nucleotides, and continue to next paragraph. 25 ml incubation medium and centrifuge immediately to sediment cells. 1 mM cAMP. Resuspend for efficient extraction of nucleotides, then continue to next paragraph.
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