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1999 Macmillan Magazines Ltd letters to nature
 

Summary: © 1999 Macmillan Magazines Ltd
letters to nature
NATURE |VOL 397 |18 FEBRUARY 1999 |www.nature.com 625
NaCl, 10 mM EDTA, 5 mM MgCl2, 1mM DTT, 1 mM GTP-gS, pH 7.5, for 90
min at room temperature under rotation. Afterwards, buffer 1 was removed
and the GTP-gS form of GST±Rab5 was stabilized with buffer (buffer 2)
containing 20 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 1mM DTT, pH 7.5, in
the presence of 1 mM GTP-gS for 20 min at room temperature under rotation.
Beads were then incubated for 120 min at 4 8C with bovine brain cytosol
obtained as follows: 14 bovine brains were homogenized in a blender with
buffer 2 and the homogenate was centrifuged at 4,200g at 4 8C for 50 min. The
resulting postnuclear supernatant was then centrifuged at 100,000g at 4 8C for
60 min. The high-speed supernatant was dialyzed against buffer 2 (without
nucleotide) before incubation with the af®nity column. After incubation with
cytosol, beads were washed with ten column volumes of buffer 2 containing 10
mM GTP-gS, ten column volumes of buffer 2 containing 250 mM NaCl ®nal
concentration and 10 mM GTP-gS, and one column volume of 20 mM HEPES,
250 mM NaCl, 1 mM DTT, pH 7.5.
Bound proteins were eluted with 1.5 column volumes of a buffer containing
20 mM HEPES, 1.5 M NaCl, 20 mM EDTA, 1mM DTT, 5 mM GDP, pH 7.5,

  

Source: Allen, John F. - School of Biological and Chemical Sciences, Queen Mary, University of London

 

Collections: Renewable Energy; Biology and Medicine