Isopropyl β d thiogalactopyranosid iptg

The R2 fragment of the gB of PCMV/PRV was purified as described^{14 (link)}. In brief, 500 mL E. coli BL21 cells containing the pET16b expression vector encoding PCMV-R2 were induced with 1 mM isopropyl-β-D-thiogalactopyranosid (IPTG) (Roth, Karlsruhe, Germany) at an OD₆₀₀ of 0.7 and cultivated at 37 °C for 2 h. Cells were harvested, centrifuged, resupended in PBS and centrifuged again. 2 g wet cells were dissolved in 10 mL 8 M urea, 0.5 M NaCl, 15 mM imidazole, 20 mM Tris pH 7.5. After a 20 min spin at 50,000 × g the supernatant is applied to a 1 mL HisTrap HP column installed on a Äkta Prime Plus system (both GE Healthcare, Chicago, Illinois, USA) using the HisTrap affinity application template with the following buffers: (1) 6 M urea, 0.5 M NaCl, 15 mM imidazole, 20 mM Tris pH 7.5; (2) 6 M urea, 0.5 M NaCl, 500 mM imidazole, 20 mM Tris pH 7.5. PCMV-R2 eluted in two peaks as was shown by polyacrylamide-gel electrophoresis. Both fractions were used for PCMV antibody detection.

Isopropyl‐β‐D‐thiogalactopyranosid (IPTG) was obtained from Carl Roth GmbH and Co. KG. Protease inhibitor cocktail set VII from Calbiochem was purchased from Merck KGaA and PLP from Sigma‐Aldrich. Immobilization supports Lifetech™ ECR were kindly donated by SpinChem AB and Purolite^®. Enzyme carrier Eupergit^® CM was purchased from Sigma‐Aldrich. D‐asparagine monohydrate was purchased from Alfa Aesar and L‐asparagine monohydrate from Sigma Aldrich chemicals, both of purity ≥99%.