By Farabee M.
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First, salt-extrated axonemes (14) are diluted (1:10) in PM buffer and centrifuged at 12,000 rpm for 10 min at room temperature (RT). The pellet is resuspended in 9 µL containing 1 mM Mg-GTP, 20 µg NEM-modified tubulin, and 20 µg PC-purified tubulin and incubated for 2 min at 37°C. The reaction is stopped by the addition of 7 µL of PM containing 40 µM Taxol (prewarmed to 37°C) and transferred to room temperature. Typically, the axoneme–MT complexes are diluted 1:5 with PM buffer before performing the assays.
In vitro motility assays are performed with the mutant proteins to check for movement on microtubules (MTs). 9 µL of 50 mM Mg–ATP. The cover slip is sealed onto a clean glass slide with VALAP and observed using video-enhanced differential interference contrast microscopy (see Note 3). Any microtubule motility is recorded on videotape. 54 Waligora and Endow 4. Once motility is acertained, polarity assays are done on each construct and compared to the wild type. The polarity assays are done the same way as the motility assays with the addition of axoneme–MTs instead of MTs.
3. The cells are chilled on ice and collected by centrifugation for 6 min at 6000g (6000 rpm in Sorvall GS3 rotor) (see Note 4). 4. The pelleted cells are stored frozen in the centrifuge bottles (see Note 5). 2. Alternate Expression for Proteins with Limited Solubility Most long kinesin constructs, as well as many constructs of other proteins, are largely insoluble when expressed at 37°C. Methods to evaluate solubility are given below. If this is a problem, then the yield of soluble protein can usually be increased by induction at a lower temperature.
ANIMAL ORGAN SYSTEMS AND HOMEOSTASIS by Farabee M.