Microseal b adhesive seal

In order to improve the stability of the purified AsFur, thermal stability in various buffer systems and salt concentrations was investigated by a Thermofluor assay (Ericsson et al. 2006 (link)). Protein unfolding and its melting temperature (T_m) is monitored by using the fluoroprobe SYPRO Orange dye which emits fluorescence upon binding to exposed hydrophobic regions.
The buffer screen contained 24 buffers covering a pH range from 4.5 to 9.0. Briefly, 5 µl protein (2.5 mg/ml), 12.5 µl 2 × buffer solution (100 mM) and 7.5 µl 300 × SYPRO® Orange (Sigma Aldrich) were mixed and added to the wells of a 48-well PCR-plate (Bio-Rad). To assess the effect of various salts, 15 µl of protein (0.8 mg/ml) diluted in the appropriate buffer (Tris pH 7.5) were mixed with 7.5 µl of 300 × SYPRO® Orange (Sigma Aldrich) and 2.5 µl different salts in concentrations ranging from 0.1–2.0 M. The plates were sealed with Microseal® 'B' Adhesive Seals (Bio-Rad) and heated in a MiniOpticon Real-Time PCR System from 20 to 80 °C in increments of 1 °C per sec. Melting curves were monitored with a charge-coupled device (CCD) camera with wavelengths for excitation and emission at 490 and 575 nm, respectively. T_m, corresponding to the midpoint of the transition curve, was determined using the supplied instrument software and monitoring the fluorescence of the HEX channel.

Triplicate first strand DNA aliquots for each sample served as templates for qRT-PCR using SoFast™ EvaGreen® Supermix (Bio-Rad, USA) on an iQ2 Optical System (Bio-Rad). Each amplification reaction was performed in a 20 µl total volume with 1 µl of cDNA and 100 nM of each primer in an iQ™ 96-well PCR plate (Bio-Rad), which was covered with Microseal “B” adhesive seals (Bio-Rad). Thermal cycling conditions included initial denaturation at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 10 s. After all reactions, a melting curve analysis from 65 to 95°C was applied to ensure consistency and specificity of the amplified product. A 10-fold dilution series of cDNA from the whole body of adults was employed as a standard curve, and the qRT-PCR efficiency was determined for each gene and each treatment with the slope of a linear regression model [44] . The corresponding qRT-PCR efficiencies (E) were calculated according to the equation: E = (10^[−1/slope] −1)×100 [45] .

Triplicate 1^st-strand DNA aliquots for each treatment served as templates for qRT-PCR using SsoFast™ EvaGreen® Supermix (Bio-Rad) on a Bio-Rad iQ2 Optical System (Bio-Rad). Amplification reactions were performed in a 20 µl volume with 1 µl of cDNA and 100 nM of each primer, in iQ™ 96-well PCR plates (Bio-Rad) covered with Microseal “B” adhesive seals (Bio-Rad). Thermal cycling conditions were as follows: initial denaturation temperature, 95°C for 30 s, followed by 40 cycles at 95°C for 5 s and 60°C for 10 s. After the reaction, a melting curve analysis from 65°C to 95°C was applied to ensure consistency and specificity of the amplified product.