Request Quote


The SDS denaturation test (SD-Test) is a Specificity Test that was developed for the analysis of the source of a Ligand-Induced Fluorescence Change that exceeds ±20% of the fluorescence average. 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 not suitable if the target is a Fluorescent fusion proteins. 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 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, repeat the centrifugation step at least 10 min at ≥15,000 g).
  • For samples containing RED-tris-NTA labeled protein, perform an ECP-Test instead of an SD-Test.


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

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 remain, 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 lab-ware.
  • Check whether the ligand exhibits fluorescence in the relevant range (Ligand Auto-fluorescence).
  • 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 the molecules of interest, by changing the pH or the ionic strength.
Return to Dianthus NanoPedia