The SDS denaturation test, or SD-Test for short, is a Specificity Test developed to analyze the source of a Ligand-Induced Fluorescence Change that exceeds ± 20% of the fluorescence mean. 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 a binding-specific effect, 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 or even increase. In case of confirmation of binding-specific changes in target fluorescence, the data can be used for interaction analysis.

Please note:

• The SD-Test is unsuitable if the target is a fluorescent fusion protein. These fluorescent proteins will also be denatured, and no fluorescence will be left for analysis.

• If potassium salts are used in the assay buffer, SDS should be avoided due to the precipitation of the salt. Instead, a final concentration of 4M Guanidine Hydrochloride should be used to denature the proteins.

• It is essential to ensure that none of the pellet after centrifuging is transferred to the new tubes. If the pellet is disturbed, centrifuge again for at least 10 min at ≥15,000 g.

• For samples containing RED-tris-NTA labeled protein or other affinity-based labeling, please perform an ECP-Test instead of an SD-Test.

Instructions

1. Centrifuge the Dianthus microwell plate for at least 10 minutes at the highest speed possible.

2. Carefully remove 15* µl of each sample and mix each with 15 µl of SD-mix (4% SDS, 40 mM DTT) in a reaction tube.

*If less than 15 µl remains, use equal volumes of supernatant and SD-mix for the test.

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

4. Load the denatured samples into fresh wells in a Dianthus microwell plate and re-measure for fluorescence intensity using, e.g., the Single-dose Quick Start experiment in DI.Control.

Recommendations in case of non-specific effects

• 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.

• Check whether the ligand exhibits fluorescence in the relevant range (Ligand Autofluorescence).

• In rare cases, the Ligand might absorb the fluorescence of the target molecule even when it is not bound (inner filter effect). In this case, lowering the ligand concentration is recommended. A control experiment for this case is explained in the article on Ligand-Induced Fluorescence Change.

• To prevent the formation of aggregates, improve buffer conditions by adding detergents or additives that stabilize your molecules, changing the pH, or changing the ionic strength.