You have observed a ligand-dependent change in initial fluorescence? In such a case performing a so-called specificity test is advisable. This article describes the observation, the underlying mechanism and the procedure in such a case. Note however, that if you used a NanoTemper Technologies RED-tris-NTA 2^nd generation labeling approach you need to perform a so-called ECP-Test instead of the SD-test described here.

If you are using MO.Control for your measurement the software can guide you through the control experiment. 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 due to 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 SD-Test as described on the next pages to determine the reasons for the fluorescence changes.

SD-Test

The SDS denaturation test, or SD-Test for short, is a specificity test that was developed for the analysis of ligand-induced changes in initial fluorescence. It helps to distinguish between fluorescence changes caused by an interaction and those caused by non-specific effects, e.g. loss of protein due to aggregation or adsorption to labware. The essential step in the protocol is the denaturation of all proteins contained in the sample using a mix of SDS and DTT in combination with heating to 95°C. Through this treatment, the target-ligand interaction will be disrupted. In case of binding-specific quenching, the fluorescence intensity in the target and complex sample will be equal after denaturation. If non-specific fluorescence loss occurs, the fluorescence intensities will remain different. In case of binding-specific changes in target fluorescence, the change in fluorescence intensity is used for binding analysis.

Refer to the MO.Control software for a detailed protocol. When ligand-specific fluorescence changes are detected in an experiment, performing the SD-Test will be recommended on the Results page. Just click the SD-Test button to start an SD-Test experiment with step by step instructions.

Please note:

• The SD-Test is not suitable if the target is a fluorescent fusion protein like GFP or YFP. These fluorescent proteins will be denatured as well, and no fluorescence will be left for analysis.
• If potassium salts are used in the assay buffer, SDS should be avoided due to precipitation of the salt. Instead, a final concentration of 4M urea should be used to denature the proteins.
• It is essential to ensure that none of the pellet is transferred to the new tubes after centrifugation. If the pellet is disturbed, centrifuge again for at least 10 min at ≥15,000 g.
• For samples containing RED-tris-NTA labeled protein, please perform an ECP-Test instead of an SD-Test.

Instructions

1. Centrifuge the remainder of tubes 1 to 3 and 14 to 16 prepared in the 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 SD-mix (4% SDS, 40mM DTT).

*Note: If less than 7 µl remain, use equal volumes of supernatant and SD-mix for the test.

3. Incubate for 5 minutes at 95°C to denature the protein.

4. Dip a Monolith NT.115 capillary into each sample and place them in the following positions of the device tray before starting the measurement:

Interpretation of SD-test results and further recommendations

Figure 1: Result of an SD-test that was run in MO.Control. The fluorescence of bound and unbound state after denaturation is equal (right), indicating that the initial fluorescence change that was observed in the original binding assay (left) was due to a binding event.

If the fluorescence intensities are identical in all capillaries after denaturation, it can be concluded that the fluorescence changes observed before denaturation were induced by a binding event. The denaturation process disrupts the binding of the ligand to the fluorescent molecule, and its initial fluorescence is restored. In this case you can analyze your data by directly using the binding information deduced from the fluorescence intensity changes.

If the differences in fluorescence intensity persist, material was lost either by aggregation and subsequent centrifugation or by nonspecific adsorption to the tube walls. In this case, the data cannot be used to extract affinities and assay conditions need to be optimized (see below).

• Add detergent to the assay buffer (0.005% Tween 20, or 0.1% Pluronic F-127) in case the ligand-induced fluorescence change is caused by adsorption to the labware or aggregation of the target.
• Use non-binding reaction tubes or MTPs to avoid adsorption of biomolecules to labware.
• 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 in the relevant range.
• In rare cases, the ligand might absorb the fluorescence of the target molecule even when it is not bound. In this case, changing target fluorescence wavelengths by using a different label is recommended. A control experiment for this case is explained in the article on ligand-induced fluorescence changes.