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GFP-BLM was purified using ion-exchange and metal chelate resin chromatography (Figure 1B).
Pooled Macroprep Q fractions containing GFP-BLM were loaded onto a 5 ml HiTrap metal chelate column (Amersham Pharmacia) charged with NiS0 4 equilibrated in Buffer B.
The metal chelate resin was washed with successive 10 column volumes of Buffer B + 10mm imidazole, Buffer B + 50 mM imidazole, and Buffer B + l00 mM imidazole.
We postulate that these enzymes are likely to chelate 2 divalent cations and are likely follow a bimetal reaction mechanism similar to what has been proposed for nucleotide cyclases, nucleic acid polymerases, or certain phosphoesterases such as those of the HD and DHH superfamilies.
CHAD contains conserved histidines that are predicted to either chelate metals or serve as phosphoacceptors.
In order to evaluate whether Btk:ER-induced calcium mobilization was due to calcium release from internal stores or calcium influx across cell membrane calcium release activated channels (CRAC), cells were pre-treated with EGTA to chelate extracellular calcium (Fig 2D, note change in scale within Fig.
The phosphorylation reaction was stopped by addition of a final concentration of 2 mM EGTA to chelate Ca 2+.
15 M EDTA to chelate Fe ions.
These structures showed that the catalytic core of the proteins consists of a double-stranded ß-helix (DSBH) fold containing a HX [DE] dyad (where X is any amino acid) and a conserved carboxy-terminal histidine which together chelate a single iron atom.
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