Full length ulp-3 was amplified from cDNA derived from adult worms using the same protocol as used in qRT-PCR experiments setion using the following oligo:5-TATATATAGGCGCGCCATGTCAGTTCCCCCGGATTTCCGC (forward) 5-GAAGCGAAATAAGGCCGGCCTATATATA (reverse)AscI was introduced at the 50 of the start codon and FseI at the 30 of the stop codon and cloned into appropriately modified pQE- 80L (His6) and pMAL-c2 (MBP). Protein expression of MBP-ULP-3 and His6-ULP-3 was performed in BL21(DE3) CodonPlus bacteria grown and induced at 37 C for 2hrs with 0.5mM IPTG. MBP-ULP-3 was purified according to manufacturer standard protocols for amylose resin (New England Biolabs). His6-ULP-3 was purified on Cobalt Talon beads (Clontech) following manufacturer instructions for soluble protein and finally dialysed three times against 50mM Tris-HCl pH7.5, 150mM NaCl, 10% glycerol, 0.5mM DTT. MBP- ULP-3 was used to immunize rabbits (BioGenes GmbH). For affinity purification of the antibodies, His6-ULP-3 was covalently coupled to AffiGel-15 resin (BioRad) and used to purify final bleeds serum.
ulp-3