ECP-Test
Have you observed a ligand-dependent change in initial fluorescence while using a NanoTemper Technologies RED-tris-NTA 2nd generation fluorophore? In such a case performing a so-called ECP-test is advisable. This article describes the observation, the underlying mechanism and the procedure.
IMPORTANT: The ECP-Test is only suited for His-tagged proteins labeled with RED-tris-NTA 2nd generation. For other types of samples, please perform the SDS-denaturation test (SD‑Test).
If you are using MO.Control for your measurement the software can guide you through the control experiments. If you however, do not have MO.Control at your disposal, use this experimental guide instead.
Ligand dependent initial fluorescence changes
If you have observed a ligand-dependent initial fluorescence change, it can occur with either of the following patterns:
What are the reasons for a ligand dependent change in fluorescence?
- Quenching or enhancement of the fluorophore’s fluorescence intensity upon binding
- Nonspecific adsorption to capillaries and/or plastic micro reaction tube walls
- 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 at capillary/tube walls or aggregation, since this causes false positive results.
If there are any indications for sticking of sample to the capillaries (split peak or shoulders of the peaks in the capillary scan), try a different type of capillary.
If there is no sticking to the capillaries or if the fluorescence changes persist even after changing to a different capillary type, please perform the EDTA/Control Peptide Test (ECP-Test) as described on the next pages to determine the reasons for the fluorescence changes.
ECP-Test
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). When ligand-induced fluorescence changes are detected in an experiment the ECP-Test will be recommended on the Results page. Just click the ECP-Test button to start the ECP-Test experiment with step-by-step instructions.
To analyze ligand-induced fluorescence changes with covalently labeled protein, perform the SD-Test instead.
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 capillaries 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.
Instructions
Prepare EDTA Test
1. Centrifuge tubes 1 to 3 and 14 to 16 prepared in your original binding assay for at least 10 minutes at ≥15.000g.
2. Carefully remove 7 μl* of each sample and mix each with 7 μl of 50 mM EDTA (pH 7.4).
3. 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 tubes containing EDTA after centrifuging. If the pellet is disturbed, centrifuge again for at least 10 min at ≥15,000 g.
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.
*Note: If less than 7 µl remain, use equal volumes of supernatant and EDTA for the test.
Prepare Control Peptide Test
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 2nd generation dye. Incubate for 30 minutes at room temperature.
2. Prepare the following assay samples:
a. Mix 20 μl of labeled control peptide with 20 μl of diluted ligand buffer for the control-peptide-only sample.
b. Mix 20 μl of labeled control peptide with 20 μl of diluted ligand for the peptide-and-ligand sample.
Load samples for both parts
Dip a Monolith NT.115 Premium Capillary into each sample and place them in the following positions of the device tray before starting the measurement:
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 can be used to extract affinities. This is only the case if both subtests show constant fluorescence in all capillaries. Extract affinities from initial fluorescence data with MO.Control (Monolith X) or MO.Affinity Analysis (Monolith NT.115, Monolith NT.115Pico, NT.Automated).
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 (0.1 % 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 ligand interaction either directly with the tris-NTA 2nd generation dye or with the His-tag. In this case, we recommend to change the labeling strategy to NHS (MO-L011 Protein Labeling Kit RED-NHS 2nd Generation – NanoTemper Technologies) or maleimide-based (MO-L014 Monolith Protein Labeling Kit RED-MALEIMIDE 2nd Generation – NanoTemper Technologies) labeling.
- To prevent the formation of aggregates, improve buffer conditions by adding detergents 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.