Step one real time pcr system

The fluorescence based melting assay was conducted using an applied Biosystems Step One Real Time PCR system and Sypro Orange dye (Sigma) [26] (link), [27] (link). A total of 10 µg of purified sdAbs was added to a 20 µl volume of PBS buffer. The Sypro Orange dye was diluted 1000-fold each into each reaction solution. The temperature was increased from 25 to 99°C at a rate of 1.2°C/min. All measurements were done in triplicate and the values agreed within 0.6°C. The fluorescence based melting measurements were also performed at least twice giving results that were essentially identical. For several sdAb clones, the melting temperature (Tm) of multiple protein preparations was measured by fluorescence based melting assay; the values determined from the different preparations agreed within a degree.

The Melting temperature was measured by circular dichroism (CD) spectroscopy. Proteins were prepared in a 1.0 cm path length quartz cuvette in 3.0 mL of water at a concentration of 20 μM. CD measurements were performed using a Jasco J-815 Spectropolarimeter equipped with a PTC-423S Peltier temperature controller. The temperature was increased from 25°C to 95°C at a rate of 2.5°C/min. Ellipticity was monitored at a wavelength of 205 nm. To monitor protein refolding, the temperature was returned to 25°C at a rate of 2.5°C/min.
The melting temperature for each of the antibodies was also determined using a thermofluor assay conducted using an Applied Biosystems Step One Real Time PCR system and Sypro Orange dye (Sigma) as described previously [20 (link)]. A total of 10 μg of each purified sdAb was added to a 20 μl final volume of PBS buffer. The Sypro Orange dye was diluted 1000-fold into each reaction solution. The temperature was increased from 25 to 99°C at a rate of 1% (about 1.2°C/min) and the fluorescence was measured via the ROX channel. All measurements were done in triplicate.