PPD2 | Protein PEAPOD 2

Application example



Equal volumes of total proteins from E. coli purified with GST agarose beads or N. benthamiana were extracted and denatured with with Laemlli buffer (50 mM Tris pH 8, 10 mM EDTA, 18% glycerol, and 10 mM DTT) at 95°C for 10 min. Samples were separated on 12% SDS-PAGE and blotted 30 min to Protran nitrocellulose membrane (Whatman) using semi-dry transfer. Blots were blocked with 0.25% BSA/0.25% gelatin for 60 min at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1000 overnight at RT with agitation. The antibody solution was decanted and the blot was rinsed briefly once, then washed 4 times for 15 min in 10mM Tris pH8.0, 0.3% Tween 20 buffer at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG alkaline phosphatase conjugated) diluted to 1:10 000 in for 60 min at RT with agitation. The blot was washed as above and developed for 10 min with NBT/BCIP (nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate; Promega Corporation).

Courtesy of Dr. Garry Sunter, South Texas Center for Emerging Infectious Disease, San Antonio, USA