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: choice of activity
* instruction of fabrics important 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
* guidance of fabrics used for examine of the cyclase
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Proper techniques for safe handling of 32Phave recently been discussed by Zoon 3 in this series. Presented below are several specific recommendations for anyone setting up this procedure. 1. Identify an isolated place, not subject to routine laboratory traffic, for the preparation. 2. Dedicate equipment such as a microfuge and automatic pipettes (Pipetman) for use with isotopes as these items are sure to become contaminated. 5 inch thick) fitted onto the barrel of a Pipetman to provide shielding to the hand while pipeting into the vial containing 32p.
Dedicate equipment such as a microfuge and automatic pipettes (Pipetman) for use with isotopes as these items are sure to become contaminated. 5 inch thick) fitted onto the barrel of a Pipetman to provide shielding to the hand while pipeting into the vial containing 32p. 3. Wear disposable surgical gowns and two pairs of gloves. The inner pair of gloves should be surgical gloves that extend up the arm to overlap 3 R. A. Zoon, this series, Vol. 152, p. 25.  PREPARATION OF Ot-32p-LABELED NUCLEOTIDES 31 the arms of the surgical gown and provide protection to the wrist.
This reaction should be done in about 15 min. 5 mM 200/zg/ml 200/,~g/ml 100 mM 25 8 16 -- 25 8 --- 25 8 -80 500 mM 2 mg/ml -- 20 mM 200 t~g/ml -- --255 400 -40 231 400 16 40 265 400 The enzymes are all from Boehringer Mannheim. The pyruvate kinase and GMP kinase are in 50% glycerol. 2 M ammonium sulfate. The ammonium sulfate is removed by centrifugation before use as described for the Stage l enzymes. not n e e d to be inactivated b e f o r e p r o c e e d i n g to Stage 4 since it eventually b e c o m e s ineffective t h r o u g h the depletion o f glucose during Stage 4.