Protoplasma

, Volume 163, Issue 1, pp 51–61 | Cite as

Isolation of F-actin from pea stems

Evidence from fluorescence microscopy
  • Shunnosuke Abe
  • E. Davies
Article

Summary

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.

Keywords

F-actin isolation Rhodamine-phalloidin Fluorescein-S1 myosin Fluorescence microscopy 

Abbreviations

CSB

cytoskeleton-isolation buffer

DTE

dithioery-thritol

EGTA

ethylene-glycol-bis(B-aminoethyl ether) N,N,N′N′-tetraacetic acid

EPPS

N-[2-hydroxyethyl]-piperazine-N′-[3-propane-sulfonic acid]

HEPES

N-[hydroxyethyl]-piperazine-N′-[2-ethanesulfonic acid]

MFSB

microfilament-stabilizing buffer

PIPES

piperazine-N,N′-bis[2-ethanesulfonic acid]

PMSF

phenylmethyl-sulfonyl fluoride

PTE

polyoxyethylene-10-tridecyl ether

TRIS

tris-(hydroxymethyl) aminoethane

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Copyright information

© Springer-Verlag 1991

Authors and Affiliations

  • Shunnosuke Abe
    • 1
  • E. Davies
    • 1
  1. 1.School of Biological SciencesUniversity of Nebraska-LincolnLincolnUSA
  2. 2.Laboratory of Biochemistry, Department of Biological Resources, College of AgricultureEhime UniversityMatsuyamaJapan

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