Isolation of F-actin from pea stems
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A procedure is introduced which allows the isolation of abundant amounts of F-actin from plants (etiolated pea seedlings) in an array of morphologies very similar to the array of morphologies found in situ. The major feature is a homogenizing medium containing very low ionic strength, low monovalent ion (K+) concentration, a 3-fold higher level of Mg+ +, the presence of EGTA to chelate Ca++, and PMSF to inhibit protease activity. Using this buffer, about 80–90% of the sedimentable actin is found in the low speed (4,000×g) pellet.
KeywordsF-actin isolation Rhodamine-phalloidin Fluorescein-S1 myosin Fluorescence microscopy
ethylene-glycol-bis(B-aminoethyl ether) N,N,N′N′-tetraacetic acid
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