By Balch W.E., Channing J.D., Hall A.
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Extra info for Small GTPases and Their Regulators Part F
25 M CaCI2. The mixture is vortexed, incubated at room temperature for 1-2 min, and added to the cells. The medium is changed on day 2 and virus is collected on day 3. 5- to 1-ml aliquots at - 8 0 ° for up to 6 months. Growth medium can also be placed on cells on day 3 for viral collection on day 4; however, titers for the second collection are much lower. In addition, virus infectivity drops with each successive freeze-thaw of stored aliquots, so storage of smaller aliquots is advisable. All solutions and plastic material in contact with virus must be bleached for at least 5 min before removal from the tissue culture biohazard hood.
Wash the column in - 5 0 ml of buffer Z plus 10-20 mM imidazole. Elute the Tat fusion protein by stepwise addition of 5-10 ml of buffer Z containing 100 mM, 250 mM, 500 mM, and i M imidazole. Analyze the start, flowthrough, and each column fraction by Coomassie blue (Fig. IC) and immunoblot analysis as outlined above. Pool appropriate fractions together. For Tat fusion proteins purified under native conditions, sonicate in 42 PROTEIN EXPRESSION AND PROTEIN-PROTEIN INTERACTIONS [9,] PBS, apply to Ni-NTA as outlined above, elute with increasing imidazole, then desalt via PD10 column into the medium of choice.
Osteoclasts were treated with the Tat leader control protein (A), Tat-HSV-TK control protein (B), or Tat-Rho V14 protein for 15 min (C) or 30 min (D). Treated cells were fixed, stained with rhodamine-conjugated phalloidin, and analyzed by fluorescence confocal microscopy. Oseoclasts treated with TatRho V14 proteins showed rapid disassembly of podosome actin structures and the appearance of stress fibers, whereas the control-treated osteoclasts maintained their podosomes. as 15 min postaddition (Fig.