ECP-Test

If a Ligand-Induced Fluorescence Change that exceeds ±20% is observed with NanoTemper Technologies RED-tris-NTA Fluorophores, performing an ECP-test is advisable. The ECP-test is a type of Specificity Test. This article describes the observation, the underlying mechanism and the procedure.

IMPORTANT: The ECP-Test is only suited for His-tag proteins labeled with RED-tris-NTA. For other types of samples, please perform the SD-Test.
What are the reasons for a ligand dependent change in fluorescence?

• Quenching or enhancement of the fluorophore’s fluorescence intensity upon binding
• Interaction of a ligand with the tris-NTA dye or the His-tag instead of the target of interest
• Nonspecific adsorption to labware
• Aggregation of the fluorescent molecule upon addition of the ligand

If ligand-dependent fluorescence changes are observed, it is necessary to rule out any material loss caused by nonspecific adsorption to labware or aggregation, since this could lead to false positive results.

Please perform the EDTA/Control Peptide Test (ECP-Test) as described here to determine the reasons for the observed fluorescence changes. The name ECP- Test is short for EDTA/Control Peptide Test. This specificity test was developed for the analysis of ligand- induced changes in initial fluorescence of His-tagged proteins, labeled with RED-tris-NTA. This test contains two subtests that must be performed to unambiguously distinguish between fluorescence changes caused by interaction and those caused by non-specific effects. In the case of His-tag labeling, non-specific effects can be caused by interaction of a ligand with the His-tag bound tris-NTA dye (Control Peptide Test) or by ligand-induced aggregation or adsorption to labware (EDTA Test).

EDTA Test

The EDTA Test helps to detect fluorescence changes caused by loss of protein due to aggregation or adsorption to labware. The high affinity of the tris-NTA dye for the His-tag is dependent on the presence of Ni(II) ions, complexed with the NTA molecule, which are complexed within the dye structure. With a high excess of the chelating agent EDTA (ethylenediaminetetraacetic acid) the Ni(II) ions are removed from the tris-NTA dye and the dye dissociates from the His-tagged protein. In case of non-specific fluorescence loss, the fluorescence intensities of the individual wells will remain different even after adding EDTA.

Please note: If the assay buffer contains divalent or trivalent cations, the concentration of EDTA has to be increased by the concentration of metal ions in the buffer to ensure the complexation of all metal ions by EDTA.

Control Peptide Test

The Control Peptide Test helps to detect fluorescence changes caused by the direct interaction of a ligand with either the tris-NTA dye or the target protein’s His-tag. The Control Peptide contains the His-tag sequence and is used instead of the target protein in a Binding Check experiment. If the same fluorescence changes are observed as for the original target, this is indicative of non-specific interactions between the tris-NTA dye/His- tag complex and the ligand.

EDTA Test Instructions

1. Centrifuge the Dianthus microwell plate for at least 10 minutes at the highest possible speed.
2. Carefully remove 10 μl* of each sample and mix each with 10 μl of 50 mM EDTA (pH 7.4) in a fresh well.

*Note: If less than 10 µl remain, use equal volumes of supernatant and EDTA for the test but make sure each well contains a final volume of 20 µl.

1. Incubate for 30 minutes at 37°C to remove the dye from the His-tag.

Note: It is essential to ensure that none of the pellet is transferred to the wells containing EDTA after centrifugation. If the pellet is disturbed, centrifuge again.
Note: If the assay buffer contains divalent or trivalent metal ions, the concentration of EDTA has to be increased by the concentration of metal ions in the buffer to ensure the chelation of all ions by EDTA.

Control Peptide Test Instructions

1. Prepare labeled control peptide sample: Label the control peptide with the tris-NTA dye by mixing 25 μl 100 nM control peptide with 25 μl 50 nM tris-NTA dye. Incubate for 30 minutes at room temperature.
2. Prepare the following assay samples:
   a. Mix 35 μl of labeled control peptide with 35 μl of diluted ligand buffer for the control-peptide-only sample.
   b. Mix 35 μl of labeled control peptide with 35 μl of diluted ligand for the peptide- and-ligand sample.
   Load samples for both parts into a Dianthus microwell plate.

Interpretation of ECP-test results and further recommendations

Taken together, the results from the EDTA Test and the Control Peptide Test determine unambiguously whether the fluorescence changes are specific to the interaction of interest and the obtained F_norm data can be used to extract affinities. This is only the case if both subtests show constant fluorescence in all wells. If the differences in fluorescence intensity persist, the data cannot be used to extract affinities and assay conditions need to be optimized:

• If fluorescence changes persist after the EDTA Test, this is indicative of material loss due to aggregation or adsorption to the labware. Add detergents to the assay buffer (0.005% Tween®20, or 0.1% Pluronic®F-127), use passivating agents (0.5 mg/ml BSA or 0.1 % PEG 8,000) and/or use non-binding reaction tubes or microtiter plates to avoid adsorption of biomolecules to labware.
• If fluorescence changes persist after the Control Peptide Test, this is indicative of a lig
  and interaction either directly with the tris-NTA dye or with the His-tag. In this case, we recommend to change the Protein Labeling strategy to NHS or maleimide-based labeling.
• To prevent the formation of aggregates, improve buffer conditions by adding deter-
  gents or additives that stabilize your molecules, by changing the pH or by changing the ionic strength.
• Check whether the ligand itself exhibits fluorescence or absorbs in the relevant
  range.