Purification of novel kinesins from embryonic systems.
Several kinesin holoenzymes, including the heterotrimeric kinesin-II and bipolar KLP61F complexes described here, are being purified in our laboratory using microtubule affinity precipitation and conventional biochemical fractionation procedures. These protocols have been optimized by using pan-kinesin peptide antibodies and subunit-specific antibodies to monitor the enrichment of kinesin-related polypeptides in particular fractions by immunoblotting. Protein purification represents the most direct route available for determining the oligomeric state and subunit composition of a kinesin holoenzyme, for identifying tightly associated accessory subunits such as SpKAP115, and for determining the molecular architecture and functional properties of native kinesin motors. Protein purification methods therefore represent an important complementary approach to molecular genetic approaches that are being pursued in many other laboratories.
Meyer
D
D
Section of Molecular and Cellular Biology, University of California, Davis 95616, USA.
Rines
D R
DR
Kashina
A
A
Cole
D G
DG
Scholey
J M
JM
D000818:Animals; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000737:chemistry; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000737:chemistry / Q000196:embryology; D004625:Embryo, Nonmammalian / Q000737:chemistry* / Q000502:physiology; D020035:Holoenzymes / Q000302:isolation & purification; D016547:Kinesins / Q000302:isolation & purification* / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000302:isolation & purification / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D020409:Molecular Motor Proteins / Q000302:isolation & purification* / Q000378:metabolism; D009124:Muscle Proteins / Q000302:isolation & purification / Q000378:metabolism; D010063:Ovum / Q000737:chemistry; D011108:Polymers / Q000302:isolation & purification / Q000378:metabolism; D012617:Sea Urchins / Q000737:chemistry / Q000196:embryology
D002135:Calcium-Binding Proteins; D029721:Drosophila Proteins; D020035:Holoenzymes; C084034:Klp61F protein, Drosophila; D008869:Microtubule-Associated Proteins; D020409:Molecular Motor Proteins; D009124:Muscle Proteins; D011108:Polymers; C074152:kinesin-II; D016547:Kinesins