Solutions of buffer and concentrated denaturant in buffer are first dispensed into Greiner Bio-One® 96-well, F-bottom, black polystyrene plates (655076) using a Microlab ML510B dispenser. We typically dispense denaturation curves in 0.1 M denaturant steps. The protein is then dispensed from a 10x concentrated stock. All plate measurements were carried out on a CLARIOstar Plate Reader (BMG Labtech) with a tryptophan detection set-up comprising two filters - an excitation filter of 295 nm and an emission filter of 360 nm - and excitation and emission bandwidths of 10 nm. The 295 nm filter is used for selective excitation of tryptophan residues. Alternatively a 280 nm filter can be used to excite both tryptophan and tyrosine residues. The high sensitivity of this state-of-the-art instrument allows readings at sub-micromolar concentrations of protein (the concentration required will, of course, depend on the number of aromatic residues in the protein and the magnitude of the fluorescence change upon unfolding).
Well volume and protein concentration are important factors for optimal signal to noise, but at the same time they will have an impact on the quantity of protein used. We find that a well volume of 150 µl is sufficient. Larger volumes will give a larger signal but will require a larger quantity of protein. We recommend a 10 µM protein stock solution, which will be diluted ten-fold to 1 µM upon dispensing. With the appropriate dispenser, the total sample volume could be further reduced to 100 µl or less.
For each protein, three sets of serial dilutions were plated consecutively. These sets can be from the same sample stock (technical replicates) or from different purifications (biological replicates). Plates were covered with a Corning® 96-well microplate aluminium sealing tape to minimise evaporation, shaken for 30 s using the CLARIOstar double-orbital shaking option, and incubated at 25 °C for 1 h. The instrument settings in the BML Labtech software were set for reading through the top orbital and “precise” rather than “rapid” measurements. Both focus height and gain were set using the fluorescence of the well having the highest fluorescence intensity. The gain adjustment was then set at 70% to prevent saturation at one or other denaturation baselines. For a good signal-to-noise ratio, we recommend to aim for a 1.5–3-fold difference in fluorescence intensity between the folded and the unfolded states.
Well volume and protein concentration are important factors for optimal signal to noise, but at the same time they will have an impact on the quantity of protein used. We find that a well volume of 150 µl is sufficient. Larger volumes will give a larger signal but will require a larger quantity of protein. We recommend a 10 µM protein stock solution, which will be diluted ten-fold to 1 µM upon dispensing. With the appropriate dispenser, the total sample volume could be further reduced to 100 µl or less.
For each protein, three sets of serial dilutions were plated consecutively. These sets can be from the same sample stock (technical replicates) or from different purifications (biological replicates). Plates were covered with a Corning® 96-well microplate aluminium sealing tape to minimise evaporation, shaken for 30 s using the CLARIOstar double-orbital shaking option, and incubated at 25 °C for 1 h. The instrument settings in the BML Labtech software were set for reading through the top orbital and “precise” rather than “rapid” measurements. Both focus height and gain were set using the fluorescence of the well having the highest fluorescence intensity. The gain adjustment was then set at 70% to prevent saturation at one or other denaturation baselines. For a good signal-to-noise ratio, we recommend to aim for a 1.5–3-fold difference in fluorescence intensity between the folded and the unfolded states.
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