LifeTein synthesized multiple peptides for the pulldown experiments. The peptides were synthesized with biotin on the N terminus and an aminohexanoic acid linker. Two types of peptides were designed: peptide without acetylation and peptide with multiple lysines (up to five sites) were acetylated in the synthetic peptides.
Pulldown Experiments With LiteTein Peptides
These peptides were bound to streptavidin-agarose beads and used for pulldown experiments using cell lysates. Peptides, streptavidin agarose beads, and cell lysates were permitted to bind for overnight at 4 °C, then beads were pelleted, washed five times using spin columns, and proteins were eluted in sample buffer and analyzed by SDS-PAGE and immunoblotting.
All synthetic peptides had an N-terminal biotin. After incubation, peptides were spotted on nitrocellulose membranes and immunoblotted to detect acetylated Lysine (AcK) or total biotinylated peptide. Data is shown below.