Pbad topo thiofusion expression kit

For the production of polyclonal antibodies, EmACT was expressed in the bacterial pBAD/TOPO ThioFusion Expression Kit (Invitrogen). To maximize the recognition of EmACT after processing by the generated antibodies, the full Emact (without stop codon) amplified using the primer pair Emact_Dw/Emact_Up coding for the preproprotein EmACT was chosen for immunization and subcloned in pBAD/TOPO ThioFusion expression vector (Invitrogen). The His-tagged Thioredoxin- fusion protein (Thio-EmACT) following arabinose induction (2 g/L; 4 h), was purified on a nickel-nitrilotriacetic acid resin (Invitrogen). The purified protein was diafiltered on against sterile PBS (1×) on Centrifugal Filter Units (Millipore) before quantification. NMRI mice were immunized with the purified Thio-EmACT resuspended in 100 μl of Freund Incomplete adjuvant (Sigma) by two subsequent sub-cutaneous injections initially and repletion 4 weeks later for boosting. Ten days after the second round of injections, animals were terminally bled from the heart and the serum was collected and stored at −20°C. To generate pre-immune serum, naïve NMRI mice were similarly bled by cardiac bleeding and serum collected.

Antibodies were raised against the intracellular domain of EmIR1 (M1129- C1749) [10 (link)]. The respective cDNA regions were amplified using primers CK5 (5′- ATC GCT GGA TCC ATA CAT CGC ATT CGA AAG AA −3′) and CK6 (5′- AAC ACA AGA TCT TGA ACA AGA CGA CCC ATC ACC GTC A −3′). The PCR product was ligated into the pBAD/Thio-Topo® vector (pBAD⁄TOPO® ThioFusion™ Expression Kit, Invitrogen, Karlsruhe, Germany) and expressed and purified according to the manufacturer’s instructions. Immunisation of a rabbit with the purified protein was performed by Immunoglobe (Himmelstadt, Germany) using program PRO-10 W-STD [48 ]. Likewise, nt sequences encoding the intracellular region of EmIR2 were amplified using primers emirbF3dw (5’-GCA ACC ACC TTC GCT AAT G-3’) and emir2-intra-up (5’-CTC AGA ATT CAT GTG AGA GTG GAA G-3’) and cloned into the pBAD/TOPO ThioFusion expression plasmid (Invitrogen). The EmIR2-Thio construct was expressed in Escherichia coli as described above for EmIR1 and the protein was purified via the His-tag. Elution fractions were dialysed and used for rabbit immunisation according to the procedure described above. In subsequent western blot analyses the purified immune-serum only detected EmIR2-Thio and EmIR2-GST, but not EmIR1-GST [see Additional file 4], thus confirming specificity.