GST-tag (mouse, monoclonal)

Proteins were isolated from E. coli Rosetta cells transformed with pGEX6P-1 vector. Lane 1 contains protein fraction from bacterial cells before induction with IPTG (-). Lane 2 contains protein fraction from bacterial cells after 2 h induction with IPTG (+). 15uL of sample were loaded and separated on an 15 % SDS-PAGE and transferred for 70 min at 55V using a tank transfer system to nitrocellulose membrane. Blots were blocked with 1x PBS + 0.1 %Tween 20 (T) + 5% milk for 1 h at room temperature (RT) with agitation. Blots were incubated with the primary antibody α-GST (AS21 4677) at indicated dilutions overnight at 4°C with agitation in 1x PBS-T + 5% milk. The primary antibody solutions were decanted, and the blots were washed 4 times (6-8 minutes each) in 1x PBS-T at RT with agitation. Blots were incubated with secondary antibody Goat anti-Mouse (Product no: AS09 627-trial) diluted to 1 : 20,000 in 1x PBS-T + 5% milk for 2 h at RT with agitation. The blots were washed as above and developed with ECL or chemiluminescent detection reagents AgriseraECL SuperBright (AS16 ECL-S-10)  for 4 min and imaged using ChemiDoc (BioRad) for 5 seconds. PonceauS served as loading control.

Courtesy of Kelly Mason and Antje Heese; Dept. Biochemistry, IPG; University of Missouri, Columbia (MO), USA