Lysine residues of bovine serum albumin (BSA) have been chemically acetylated by acetic anhydride under basic conditions. A major Ac-BSA band runs at approximately 66 KDa and Ac-BSA oligos run at approximately 120-260 KDa. NOTE: Ac-BSA oligomerization is not caused by the acetylation reaction as these species are also present in unmodified BSA as seen in a Coomassie stain of unmodified BSA protein. Acetylated BSA is supplied as a lyophilized powder.
Figure 1: Titration of Ac-BSA to determine antibody specificity and sensitivity
Legend: Lanes 1-5, 0.5, 0.1, 0.05, 0.01 and 0.005 ng Ac-BSA respectively. Lanes 6 & 7, 500 and 1000 ng of non-acetylated BSA. Western blots of Ac-BSA and unmodified BSA were ana-lysed using AAC01 anti-acetyl antibody. The 1000 ng of non-acetylated BSA did not produce a signal while Ac-BSA was de-tected down to 0.005 ng. The major BSA band runs at approxi-mately 66KD (arrowhead) with BSA oligomers running between approximately 120-260 KDa.
Figure 2: Ac-BSA competition for antibody binding to acetyl lysine targets
Legend: Lysates were made from A431 cells that were treated (+) or untreated (-) with the HDAC inhibitor trichostatin A (TSA). Each lane represents 20 µg of cell lysate. Lysates were run on SDS-PAGE and transferred to PVDF membranes. Membranes were probed with anti-acetyl lysine antibody (Cat. # AAC01) at 1:500 dilution in TBST in the presence of 10 µg/ml BSA or 10 µg/ml of Ac-BSA. The Ac-BSA eliminates all acetyl lysine signal on the membrane. Prominent bands in the TSA treated lanes represent acetyl tubulin (55 KDa) and acetyl histones (14-16 KDa).
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