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View additional product information for Pierce™ Nickel Coated Plates - FAQs (15142, 15442, 15342, 15242)
12 product FAQs found
The binding capacity for Pierce Nickel Coated Plate is approximately 9 pmol His-tagged protein (27 kDa) per well.
Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.
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).
The Pierce Nickel Coated Plates are extremely stable over a long period of time, if stored at room temperature (20-25 degrees C). We guarantee 12 mths of shelf life in unopened foil package after the date of shipment.
Yes. When setting-up the assay, the concentration of the His-tagged protein/peptide should be determined by preparing a titration. We recommend using a solution of the His-tagged fusion protein/peptide diluted in KCl, in a concentration ranging from 0.01-10 µg/mL. Hereafter, the primary and secondary antibodies should be optimized according to the manufacturer's recommendations.
We recommend pre-washing Pierce Nickel Coated Plates with PBS containing 0.05 % (v/v) Tween-20 surfactant to saturate the plate surface. This minimizes the non-specific binding during the assay and provides a low background.
Yes. The interaction is pH-dependent, and binding is best obtained at neutral pH or slightly above neutral pH. We recommend using 10 mM KCl, which has a neutral pH, as a coupling buffer.
Due to steric hindrance, optimal performance is normally observed if the fusion protein contains more than four neighboring histidine amino acids located at the terminal end. In some cases the histidine amino acids do not need to be placed next to each other - just be sure that the 3D structure fold them next to each other (e.g., the HAT fusion protein).
The Pierce Nickel Coated Plates can via a simple one-step protocol bind all types of His-tagged fusion proteins/peptides. This includes purified as well as crude cell lysates containing His-tagged fusion proteins/peptides.