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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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