Method for detecting acetylated PD-L1 in cell lysates v1

Here we present a method that allows detection of acetylated PD-L1 and is applicable to a wide range of cell lines. The method captures >90% of acetylated PD-L1 species, is semi-quantitative and simple to perform in any lab equipped with tissue culture and western blot equipment. The method involves processing cells in a lysis buffer that has been optimized for efficient immunoprecipitation (IP) of acetylated species, an IP enrichment step utilizing an acetyl-lysine affinity matrix and western blot detection of both total and acetylated PD-L1 on the same blot. This technique compliments the alternative IP approach utilizing a PD-L1 antibody as the IP reagent and an anti-acetyl lysine antibody as the detection reagent. However, because the protocol described here enables the detection of both total and acetylated PD-L1 on the same blot, this method has the advantage of allowing quantitation of the percent of PD-L1 that is acetylated, an important parameter for mechanistic interpretation. The method described here utilizes beads that are covalently linked to the affinity antibody, resulting in extremely clean IP results. Western blots can be re-probed with a pan anti-acetyl lysine antibody to visualize the total protein acetylation profile in any given lysate, a property that is useful when examining PD-L1 acetylation in the presence of HDAC inhibitors or other treatments affecting global acetylation.