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.
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.
Acetylation of TUG Protein Promotes the Accumulation of GLUT4 Glucose Transporters in an Insulin-Responsive Intracellular Compartment, The Journal of Biological Chemistry, January 5, 2015, doi: 10.1074/jbc.M114.603977 jbc.M114.603977.