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Catalog Number | Color | Format | Detection Method |
---|---|---|---|
15142 | Clear | Strip Plate | Colorimetric |
15342 | Black | Standard | Fluorescence |
15442 | Clear | Standard | Colorimetric |
15242 | White | Standard | Chemiluminescence |
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The water used for washing of microplates as well as all assay reagents, must be of absolutely ultra-pure quality. This means that no metal ions should be present in the water, as this will bind to the His-tag and thereby decrease the binding of the fusion protein to the nickel-chelate complex.
Yes. For instance, use imidazole in concentrations >500 mM in Tris at pH 7.5 or a high concentration of EDTA.
Ionic detergents (e.g., SDS) will interfere with the binding as well as high concentrations of chelating reagents like EDTA, EGTA, and very strong electron donors like metal ions.
To determine the approximate amount of bound His-tagged fusion protein/peptide, a standard curve of a previously purified preparation can be applied.
The detection limit for each protein depends on the assay system used (e.g., primary and secondary antibody, incubation time, detection reagent), the accessibility of the His-tagged fusion biomolecule, and the size of the protein (large proteins bind with a low density). Using a 25 kDa 6xHis-tagged protein, we have observed a detection limit of 1.0 ng per well (100 µL).
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