Could anybody tell me about the principle of cushion buffer for microtubule sedimentation? I has established an in vitro microtubule reassembly system from sperm axoneme. Although a typical sigmoid curve of tubulin assembly could be observed in turbidimetric assays, I still coud not recover reassembled microtubules by ultracentrifugation. I didn't use cushion buffer to sediment microtubules but the pellet is actually visible. However, SDS-PAGE analysis is totally smear. Is cushion buffer essential? Does ultracentrifugation crush reassembled microtubules permanently so that I can't resolve reassembled microtubules in SDS-PAGE? If cushion buffer is essential for sedimentation assays, how many volume ratios and density shoud be adequate to pellet microtubules?
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Posted 11 July 2010 - 12:38 PM
Could anybody tell me about the principle of cushion buffer for microtubule sedimentation? I has established an in vitro microtubule reassembly system from sperm axoneme. Although a typical sigmoid curve of tubulin assembly could be observed in turbidimetric assays, I still coud not recover reassembled microtubules by ultracentrifugation. I didn't use cushion buffer to sediment microtubules but the pellet is actually visible. However, SDS-PAGE analysis is totally smear. Is cushion buffer essential? Does ultracentrifugation crush reassembled microtubules permanently so that I can't resolve reassembled microtubules in SDS-PAGE? If cushion buffer is essential for sedimentation assays, how many volume ratios and density shoud be adequate to pellet microtubules?
