Fluorescence quenching assay

Measuring fluorescence quenching using capillary scanning function of the Monolith NT.115 Instrument

Set-up for this assay was adapted from the MST starting guide by Nanotemper Technologies.

1. Prepare 2-fold dilutions of 2x unlabeled peptide into 20 mM HEPES pH 7.2, 100 mM KOAc, 0.1% Tween-20, 1% DMSO
2. Distribute 10 ul of 2x unlabeled peptide into PCR tubes
3. Prepare working stock of 400 nM AlexaFluor647-labeled ECD protein in 20 mM HEPES pH 7.2, 100 mM KOAc, 0.1% Tween-20
4. Add 10 ul of the labeled ECD protein for a final concentration of 200 nM
5. Incubate samples for 30 min at RT in the dark
6. Load samples by capillary action onto Premium capillaries (Nanotemper Technologies, cat. #MO-K025).
7. Scan for fluorescence on a Monolith NT.115 Instrument (NanoTemper Technologies, Germany) at 25°C, medium power.

Important factors to avoid non-specific fluctuations in fluorescence:

• Keep % of DMSO constant throughout all samples
• Use 0.1% Tween-20 in the final sample to prevent aggregation of peptide
• Make serial dilutions from a singular working stock of peptide
• Use Premium capillaries (#MO-K025)

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