Pmal cri plasmid

Full-length cDNA of Mst35Bb was obtained by PCR from cDNA library of y w testicular total RNAs. Mst35Bb cDNA with EcoRI and XhoI restriction site was amplified using a primer set (5′-CGGAATTCATGAGTTCAAATAATGTAAATGAGTGC-3′, 5′-CCGCTCGAGTTACTTGCAAATCCGTCG-3′), and cloned into the pGEX-4T-1 plasmid (GE Healthcare) in EcoRI and XhoI site, and the pMAL-cRI plasmid (New England Biolabs) in EcoRI and SalI site. The following steps were performed as anti-Mst77F [FL] antibody generation.

Full-length cDNA of Mst77F was obtained from the Drosophila Gene Collection (DGC) (Clone ID #RH09844). Mst77F cDNA was amplified using a primer set (5′-CGGAATTCATGAGTAATCTGAAACAAAAGG-3′, 5′-AGCTCGAGTTACATCGAGCACTTGGGCTTG-3′), and cloned into the EcoRI and XhoI sites of the pGEX-4T-1 plasmid (GE Healthcare), and into EcoRI and SalI sites of the pMAL-cRI plasmid (New England Biolabs). These plasmids were both transformed into E. coli BL21-CodonPlus (DE3)-RIL (Agilent Technologie). First, E. coli was incubated at 37°C for 3 h, and after adding 1 mM isopropyl beta-d-thiogalactoside (IPTG), incubated at 30°C for more 3 h. Cells were harvested, and the lysis buffer (20 mM Tris-HCL pH 8.0, 150 mM NaCl, 0.1 mM EDTA, 1% Trition X) was added, and briefly sonicated. After centrifugation, the supernatant was collected as a soluble fraction. For glutathione S-transferase (GST) fused Mst77F recombinant protein, the soluble fraction was incubated with glutathione sepharose 4B (GE Healthcare) and eluted by glutathione, and dialysed with 1× PBS buffer, and used as an antigen for the rabbit polyclonal antibody. For maltose-binding protein (MBP) fused Mst77F recombinant protein, the soluble fraction was incubated with amylose resin (New England Biolabs) and eluted by maltose, and dialysed with 1× PBS buffer, and used for the affinity purification.