Mj minicycler

Fluorescence-based protein thermostability was determined for OXA-48, OXA-48:P68A, and OXA-48:P68A,Y211S in an MJ minicycler (Bio-Rad) across a temperature gradient of 25 to 60°C (at a heating rate of 1°C per min). Thermostability was determined in 50 mM HEPES (VWR, PA) at pH 7.5 supplemented with 50 mM potassium sulfate (Honeywell, NC) using 0.2 mg/ml protein and 5× SYPRO orange (Sigma-Aldrich, St. Louis, MO). The excitation and emission wavelengths of SYPRO orange are 470 and 570 nm, respectively. The melting temperatures were determined as the inflection point of the melting transition found from the first derivative. All experiments were performed in triplicates.

Thermostability was performed as previously published using purified OXA-48 enzymes, containing 6-His-tag and TEV cleaving site^{23 (link)}. In 50 mM HEPES (VWR), pH 7.5 including 50 mM potassium sulfate (Honeywell), enzymes were diluted to 0.2 mg ml⁻¹ and mixed with 5xSYPRO orange (Sigma-Aldrich). Using an MJ minicycler (Bio-Rad), a temperature gradient (25 °C to 70 °C) was performed with a heating rate of 1 °C min⁻¹. All experiments were performed in triplicates, and the melting temperatures (T_M) were determined as the inflection point of the melting transition found from the first derivative.

Reverse transcription and amplification was performed on pooled NEB ME and CAE samples, and on samples of whole lung tissue by the Ovation PicoSL WTA System V2 kit (NuGEN Technologies, Leiden, The Netherlands) on an MJ Mini Cycler (Bio-Rad, Temse, Belgium) starting from picograms of total RNA. Amplification can start randomly throughout the whole transcriptome in the sample, making the protocol ideal for amplification of short strands of RNA. First, single stranded cDNA is prepared, resulting in a cDNA/mRNA hybrid molecule with a unique RNA tag sequence (SPIA tag) at the 5 ´ end of the cDNA strand. Next, fragmentation of the mRNA within the cDNA/mRNA complex initiates synthesis of a second cDNA strand after adding DNA polymerases, and creates a double-stranded cDNA with a DNA/RNA heteroduplex corresponding to the SPIA tag at one end. Adding DNA polymerases starts the synthesis of cDNA at the 3′ end of the primer, removing the existing forward strand. Repetition of this process, by using a defined program of consecutive thermal steps for the SPIA DNA/RNA primer binding, DNA replication, strand displacement and RNA cleavage, results in a rapid amplification of cDNA.

The thermal stability of TfSOD was determined using a fluorescence-based thermal stability assay in an MJ minicycler (Bio-Rad) [47 (link)]. The assay volume used was 25 µl, which included (final concentrations) 0.50 mg/ml enzyme, 300X SYPRO Orange solution from a 5000X stock solution, and 100 mM buffers at pH 5.0 (Sodium acetate), pH 6.0 (MES), pH 7.0 (Tris–HCl), pH 8.0 (Tris–HCl), pH 9.0 (Bicine), and pH 10.5 (CHAPS). The temperature gradient was from 10 to 95 °C with an increase of 1 °C per minute. The melting temperature (T_m) was determined to be the inflection point of the melting transition found from the first derivative. All experiments were performed in duplicate.
The principles behind the fluorescence-based thermal stability assay are that the fluorescence dye (SYPRO Orange) binds the hydrophobic residues that gets exposed during unfolding and give the fluorescence signal. At temperatures higher than the fluorescence peak, the protein aggregates, and the fluorescence signal drop due to lack of dye to protein interactions.