Isolation of F-actin from pea stems |
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Authors: | Shunnosuke Abe E Davies |
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Institution: | (1) School of Biological Sciences, University of Nebraska-Lincoln, 68588-0118 Lincoln, NE, USA;(2) Present address: Laboratory of Biochemistry, Department of Biological Resources, College of Agriculture, Ehime University, Tarumi, Matsuyama, Japan |
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Abstract: | 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.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 -bis2-ethanesulfonic acid]
- PMSF
phenylmethyl-sulfonyl fluoride
- PTE
polyoxyethylene-10-tridecyl ether
- TRIS
tris-(hydroxymethyl) aminoethane |
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Keywords: | F-actin isolation Rhodamine-phalloidin Fluorescein-S1 myosin Fluorescence microscopy |
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