Hitrap nhs activated high performance column

Antibodies were raised against a maltose-binding protein (MPB) containing a specific region of the 3rd cytoplasmic loop (CPL3) of PeaDOP2 (see Figure 1), which was constructed as follows. The CPL3-specific fragment was amplified by PCR with AB-F (5′-AAAGAATTCGCAGTAAT CCAGACG-3′) and AB-R (5′-TTTGTCGACTCAGTGATGTGGAGGAGACAT-3′) as the sense and antisense primer, respectively. The fragment was cloned into the pMAL-c2X vector (New England Biolabs, Frankfurt, Germany). The fusion protein was over-expressed in E. coli BL21 and purified by amylose affinity-chromatography. Anti-PeaDOP2 polyclonal antiserum was raised in rabbits (Pineda-Antikörper-Service, Berlin, Germany). For purification of crude anti-PeaDOP2-antiserum, the CPL3-specific fragment of PeaDOP2 was cloned into the pET-30a vector (Novagen, Darmstadt, Germany) and over-expressed. The purified protein was coupled to a HiTrap NHS-activated High Performance column (GE Healthcare, Freiburg, Germany). Antibodies from 50 mL crude antiserum were affinity purified as described previously [60 (link)].

All proteins used in this study were produced essentially as previously outlined, i.e. hHDM-FH (53 (link)). Briefly, for mHDM-FH, a single stable expressing clone was selected for protein production and scaled up in static tissue culture before transfer to roller bottles for 10 days (in the absence of Hygromycin B). Cell debris was removed by centrifugation and supernatant was sterile-filtered prior to loading on a 2 ml HiTrap NHS-activated high-performance column (GE Healthcare), which had been previously coupled (following manufacturers guidance) with the anti-mFH monoclonal antibody 2A5 (gift from Prof C. Harris, Newcastle UK), using an AKTA START (GE Healthcare) at a flowrate of 1 ml/min. Washes of 10 column volumes were performed prior to elution. Elution was achieved using 0.1M Glycine (pH of 2.7) and eluate was collected into 1M Tris pH 9.0. All protein-containing fractions were then combined and buffer exchanged into PBS using a PD-10 column (GE Healthcare). Proteins were further polished using a gel filtration column (Superdex 600, GE Healthcare) equilibrated with PBS buffer with an ÄKTA Purifier set to a flow rate of 0.5ml/min. One ml fractions were collected and stored at -80°C until needed.