Isolating tubulin from nonneural sources.
Tubulin is a highly conserved, negatively charged protein that is found in essentially all eukaryotic cells. These properties ensure that isolation protocols successful in one system will likely work, with a few modifications, in most systems. Tubulin has been isolated most frequently from mammalian brain, and the main difference encountered in other systems versus brain is that tubulin is much less abundant in nearly all other sources than it is in brain. This means that attempting to purify tubulin by direct polymerization from a homogenate will often fail or be quite inefficient. However, the conservation of negative charge on tubulin means that an initial ion exchange step can be used to both purify and concentrate the protein from most systems. Polymerization-competent tubulin can usually be obtained by inducing polymerization in the salt eluate from the ion exchange step. We describe protocols for this procedure and describe its application to a number of vertebrate, fungal, protozoal, and plant sources.
Sackett
Dan L
DL
Laboratory of Integrative and Medical Biophysics, Program in Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
Werbovetz
Karl A
KA
Morrissette
Naomi S
NS
D000818:Animals; D002477:Cells / Q000737:chemistry* / Q000378:metabolism; D019411:Clinical Laboratory Techniques; D006801:Humans; D016296:Mutagenesis / Q000502:physiology; D009474:Neurons / Q000737:chemistry / Q000378:metabolism; D010271:Parasites / Q000737:chemistry / Q000378:metabolism; D010944:Plants / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000378:metabolism; D016808:Tetrahymena thermophila / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014404:Tubulin / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D014404:Tubulin