Anti ddk monoclonal antibody

HEK293 cells were transfected with 5 μg of (Myc-DDK-tagged)-Human CD59 plasmid (OriGene) and 2.5 μl of EndoFectin Plus transfection reagent (GeneCopoeia) in a confluent 35-mm plate according to the manufacturer's protocol. After transfection for 72 hours, the HEK293 cells were lysed with immunoprecipitation (IP) lysis buffer (Thermo Scientific). The expression of Myc-DDK–tagged human CD59 was confirmed by detecting DDK with anti-DDK monoclonal antibody (OriGene) by Western blotting.

For Western blotting assays, the cells were lysed on ice in radioimmunoprecipitation buffer (0.05 M Tris-HCl [pH 7.4], 0.15 M NaCl, 0.25% deoxycholic acid, 1% IGEPAL CA-630, and 1 mM ethylenediaminetetraacetic acid). The lysates were centrifuged at 13,000 rpm at 4 °C for 10 min. The protein extracts were solubilized in sodium dodecyl sulfate (SDS) gel loading buffer (60 mmol/L Tris base, 2% SDS, 10% glycerol, and 5% β-mercaptoethanol). Samples containing equal amounts of protein (40 μg) were separated on an 8% SDS-polyacrylamide gel by using electrophoresis and electroblotted onto Immobilon-P membranes (Millipore, Bedford, MA, USA) in transfer buffer. Immunoblotting was performed using an anti-DDK monoclonal antibody (1:3,000, OriGene, Rockville, MD, US) that was raised against the Myc-DDK-hTMEM240 protein. β-actin (1:5,000, GeneTex, Texas, USA) was used as an internal control.