AlphaScreen Histidine (Nickel Chelate) Detection Kit for HTS Assays

April 27, 2017

University of Kansas Medical Center
Pathology
Postdoctoral Fellow

Overall

Quality of Results

Ease-of-Optimization

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Company:

Revvity

Product Name:

AlphaScreen Histidine (Nickel Chelate) Detection Kit

Catalog Number:

6760619

This assay was used to screen for novel inhibitors of a protein-protein interaction. We used a His-tagged recombinant protein (protein A), coupled to these beads. We also used the GSH beads (separate review), with a GST-tagged recombinant protein (protein B). We were able to identify compounds that may interfere with binding of protein A to protein B.

Experimental Design and Results Summary

Application

AlphaScreen HTS Assay

Starting Material

Human recombinant proteins

Protocol Overview

Following optimization of buffer, salt concentration, order of reagent addition and concentration of reagents, the beads, recombinant proteins and drug compound of interest were mixed and incubated for 2 hours at RT in 96 well plates. Reactions were read using a Perkin Elmer Enspire using AlphaScreen default label every hour up to 8 hours to study effects of the drug compound on AlphaCounts.

Tips

Carefully optimize assay conditions, particularly order of addition of reagents/proteins and buffer composition.

Results Summary

We were able to identify compounds that disrupted binding of our proteins of interest at a high level compared with background.

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

This assay makes HTS assays achievable in academic research labs.

The Bad

Assay reagents are costly, espescially during the optimization phase.

The Bottom Line

The AlphaScreen kit allows academic scientists to perform high quality drug screening assays in their laboratory. Consideration should be made for drugs that may have non-specific effects in a nickel-based screen.

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