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n-EEEE-, where X is any amino acid and n = 0, 1, or 2) is 158, which is much larger than that of the corresponding oligo-aspartate sequence. This is also an advantage of the oligo-aspartate tag.
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The binding between 1-2Zn(II) (25 μM) and the D4 peptide was also evaluated by ITC under slightly acidic conditions (50 mM MES buffer, pH 5.5). Heat evolution due to the complex formation ceased sharply after the addition of 1.5 equiv of the D4 peptide. This suggests that the binding affinity of 1-2Zn(II) for the D4 peptide is partially maintained under the acidic conditions, though detailed thermodynamic analysis cannot be done because the resultant binding isotherm is biphasic.
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The in vitro fluorescence titration experiment confirmed that the fluorescence intensity of Cy5-3-4Zn(II) scarcely changed upon binding to the D4-G-D4 peptide.
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33747602894
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Since FITC-2-4Zn(II) and Cy5-3-4Zn(II) are not membrane-permeable, they are useful for the labeling of cell-surface membrane receptor. On the other hand, we found that the fluorescent DpaTyr dimers, having a BODIPY or a rhodamine derivative, can penetrate through the cell membrane so that these derivatives localize in the cytoplasm of CHO cell.
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