Rotor gene q 2plex hrm qpcr cycler

A fluorescence-based procedure for the assessment of detergent-solubilised and purified membrane protein stability was used, following established methods26 (link)28 (link). Briefly, 5 mg mL⁻¹ stocks of 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) dissolved in DMSO were diluted 50-fold into the assay buffer (20 mM tris-HCl pH 7.4, 20 mM NaCl, 0.1 mg mL⁻¹ tetraoleoyl cardiolipin, 0.1% (w/v) lauryl maltose neopentyl glycol) and incubated for 10 min at room temperature. For each test, purified protein was diluted 50-fold in the same assay buffer (approximately 50 μg mL⁻¹), and incubated on ice for 10 min in 200 μL thin-walled PCR tubes. 5 μL of the CPM dilution was added to the sample to make a final volume of 50 μL and incubated on ice for a further 10 min. The samples were subjected to a high-resolution melt (HRM) procedure on a Rotor-Gene Q 2plex HRM qPCR cycler with a 36-sample rotor (Qiagen, Venlo, the Netherlands).
In the assay, buried cysteine residues become solvent-exposed as protein unfolds, and react with CPM to form fluorescent adducts (ex/em optima of 387/463 nm). Protein unfolding profiles were analysed using the Rotor-gene Q software and the peak in the derivative of the fluorescence signal as a function of temperature, the ‘melt’ temperature (T_m), provided a relative measure of protein stability.

A microscale method, based on the thiol reactive fluorophore N-[4-(7-diethylamino-4-methyl-3-coumarinyl)-phenyl]-maleimide (CPM), was used to measure the thermostability of SLC25A25 [62 (link),63 (link)]. CPM dissolved at 5 mg/ml in DMSO was diluted into assay buffer (purification buffer B) and incubated at room temperature in the dark for 10 min. The protein stock was diluted to 40 μg/ml in the assay buffer (±1 mM CaCl₂) and incubated on ice for 10 min. CPM working solution (5 μL) was added, and the sample was vortexed briefly and incubated at 4 °C in the dark for a further 10 min. Data were collected using a high-resolution melt (HRM) channel on a RotorGene Q 2plex HRM qPCR cycler with a 36-sample rotor (Qiagen). Measurements were made every 15 s at 1 °C intervals from 25–90 °C. Protein denaturation profiles were analyzed using the Rotor-gene Q software, and the peak in the derivative of the fluorescence was plotted from which the apparent melting temperature (T_m), a relative measure of protein stability, was determined.