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.
- 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.