Abstract
Publisher Summary High-pressure liquid chromatography (HPLC) on a Mono Q column has been used to separate various axonemal dyneins. This procedure takes only an hour or two. It has the further advantage that it is able to separate multiple inner-arm dyneins, which sucrose gradient centrifugation is unable to separate; however, the sample obtained by Mono Q chromatography may be contaminated by proteins that have similar charges. Therefore, it is recommended that sucrose gradient centrifugation be used in combination with Mono Q chromatography when very pure samples are desired. The procedure described in this chapter is based on the works of Goodenough and associates and has been used to separate seven discrete subspecies of inner-arm dyneins from Chlamydomonas axonemal extract. There is description of the solutions needed and the procedures of dynein extraction and mono Q chromatography. Most studies on isolated dynein have used a purification step with sucrose density gradient centrifugation. Although this method can separate dynein from contaminants having low sedimentation coefficients, it has a significant disadvantage that the centrifugation process takes as long as 5–8 hours. The HPLC method described in the chapter is preferable.
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