By Nathan P. Kaplan, Nathan P. Colowick, Jackie D. Corbin, Joel G. Hardman
The severely acclaimed laboratory ordinary, Methods in Enzymology, is without doubt one of the such a lot hugely revered courses within the box of biochemistry. due to the fact that 1955, every one quantity has been eagerly awaited, usually consulted, and praised via researchers and reviewers alike. The sequence includes a lot fabric nonetheless appropriate at the present time - actually an important booklet for researchers in all fields of existence sciences
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Extra resources for Hormone Action Part F: Protein Kinases
The fractionation is allowed to proceed for 6-8 sec to ensure release of 90-95% of the cytoplasmic proteins and then the centrifuge is run for 30 sec. When the centrifuge stops, the supernatant proteins and/or the cell pellet are removed from the Eppendorf tube and prepared for electrophoresis as described below. The tube is discarded in the radioactive waste. Preparation o f Cells without Fractionation. A number of investigators have elected not to fractionate the cells before preparing the samples for 1o A.
78, 6903 (1981). k S. A. Rudolph and P. Greengard, J. Biol. Chem. 255, 8534 (1980). t W. B. Benjamin and I. Singer, Biochemistry 14, 3301 (1975). m J. Avruch, G. R. Leone, and D. B. Martin, J. Biol. Chem. 251, 1511 (1976). n j. Avruch, L. A. Witters, M. C. Alexander, and M. A. Bush, J. Biol. Chem. 253, 4754 (1978). o j. C. Garrison, J. Biol. Chem. 253, 7091 (1978). P T. M. Koroscil and S. Gallant, J. Biol. Chem. 256, 6700 (1981). 22 GENERAL METHODOLOGY  nique has been used. The sections below present protocols for performing these experiments based on the author's experience with isolated hepatocytes.
K. Bor|and, V. A. Florio, and D. A. Twible, J. Biol. Chem. 254, 7147 (1979). ~5j. Avruch, G. R. Leone, and D. B. Martin, J. Biol. Chem. 251, 1505 (1976). 16j. Avruch, G. R. Leone, and D. B. Martin, J. Biol. Chem. 251, 1511 (1976). J7 j. E. B. Fox, A. K. Say, and R. J. Haslam, Biochem. J. 184, 651 (1979). 26 GENERALMETHODOLOGY  ommend any one scheme for maintaining the phosphorylation state of individual proteins while fractionating cells. Each tissue should be approached on an individual basis.
Hormone Action Part F: Protein Kinases by Nathan P. Kaplan, Nathan P. Colowick, Jackie D. Corbin, Joel G. Hardman