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Isolation of F-actin from pea stems

Evidence from fluorescence microscopy

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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.

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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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Author notes

  1. Shunnosuke Abe

    Present address: Laboratory of Biochemistry, Department of Biological Resources, College of Agriculture, Ehime University, Tarumi, Matsuyama, Japan

Authors and Affiliations

  1. School of Biological Sciences, University of Nebraska-Lincoln, 68588-0118, Lincoln, NE, USA

    Shunnosuke Abe & E. Davies

Authors
  1. Shunnosuke Abe
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  2. E. Davies
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Abe, S., Davies, E. Isolation of F-actin from pea stems. Protoplasma 163, 51–61 (1991). https://doi.org/10.1007/BF01323406

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  • DOI: https://doi.org/10.1007/BF01323406

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