A pH- and temperature-dependent cycling method that doubles the yield of microtubule protein.
This report describes a procedure for the isolation of microtubules (MTs) and microtubule-associated proteins from mammalian cultured cells and tissues. This method relies on the discovery that the solubility of brain tubulin is pH-sensitive. In this report, we examined tubulin solubility over a broad pH range and discovered that the amount of soluble tubulin increased by 240% as the pH was raised from 6.0 to 8.0. Quantitative immunoblotting revealed that there was an almost equal partitioning of tyrosinated and acetylated alpha-tubulin isotypes at the higher pH. These observations were incorporated into a new procedure for the purification of microtubule protein (MTP) from bovine brain and mouse B16 cultured melanoma cells. Morphologically, and in terms of polypeptide composition, this MTP is indistinguishable from that prepared by a glycerol-cycling method (21). Moreover, our new method of pH- and temperature-cycling yields almost twice as much MTP as that obtained by other cycling methods.
Tiwari
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SC
Department of Physiology and Cell Biology, University of Kansas, Lawrence 66045.
Suprenant
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KA
D000107:Acetylation; D000818:Animals; D001923:Brain Chemistry; D002021:Buffers; D002417:Cattle; D002623:Chemistry Techniques, Analytical / Q000379:methods; D002627:Chemistry, Physical / Q000379:methods; D006863:Hydrogen-Ion Concentration; D008546:Melanoma, Experimental / Q000737:chemistry; D051379:Mice; D008868:Microtubule Proteins / Q000302:isolation & purification*; D008870:Microtubules / Q000737:chemistry; D012995:Solubility; D013696:Temperature; D014404:Tubulin / Q000737:chemistry / Q000302:isolation & purification; D014443:Tyrosine / Q000737:chemistry
D002021:Buffers; D008868:Microtubule Proteins; D014404:Tubulin; D014443:Tyrosine