AAC03, anti-acetyl lysine antibody is a pan-acetyl lysine mouse monoclonal antibody that is part of the Signal-Seeker™ product line.The Anti-acetyl-lysine antibody recognizes proteins post-translationally modified by acetylation on the epsilon amine groups of lysine residues that occur on 30-50% of all proteins and in particular histones, p53, tubulin and myosin. A proprietary mixture of acetylated proteins was used to produce a highly robust antibody that has been shown to recognize a wide range of acetylated proteins in IP, WB, ChIP and IF applications. This Anti-acetyl-lysine antibody has many advantages when compared to other commercially available antibodies as shown below.
Western Blot using Acetyl-Lysine Antibody (AAC03)
Fig 1: A: Murine tissue extract, 30 μg brain extract. B: 30 μg of Cos-7 cell lysate treated with TSA and nicotinamide (+) or untreated (-). Strongly enhanced bands at 55 and 14-16 kDa in TSA-treated lysate correspond to acetylated tubulin and histone proteins, respectively. C: Titration of acetylated BSA. Lanes 1-5 contain 0.5, 0.1, 0.05, 0.01, and 0.005 ng Ac-BSA, lanes 6-7 contain 500 and 1000 ng non-acetylated BSA, respectively. AAC03 was used at a 1:500 dilution following the recommended western blot protocol.
Immunoprecipitation using Acetyl-Lysine Antibody
Fig 2: Cos-7 cells were either treated (+) or untreated (-) with TSA (1 μM) and nicotinamide (1 mM) for 6 hours. Cell lysates were prepared in BlastR buffer and filter system and 1 mg of lysate per reaction was used for IP of acetylated proteins. 20 ml of AAC03 was used per IP reaction. Western blots of immunoprecipitated proteins were developed using AAC03-HRP at 1:3000 dilution.
Fig 3: Swiss 3T3 cells, untreated (a and c) or treated (b and d) with TSA (1 μM for 6 h), were stained as described in the datasheet. Acetylated proteins were visualized using a green fluorescent secondary. Actin fibers were visualized using a red Rhodamine Phalloidin and the nucleus was stained with DAPI. The acetylated microtubule network is clearly visible with TSA-treatment, while the green fluorescent nuclear intensity indicate the high abundance of acetylated proteins in the nucleus. In c and d, acetylated BSA (10 mg/ml) was used to compete for AAC03 binding as an indicator of AAC02 specificity for acetyl-lysine modifications.
Fig 4: Utilization of AAC03 for ChIP. Chromatin was prepared from A431 cells, either untreated or treated with TSA (1 μM) and nicotinamide (1 mM) for 6 hours. ChIP was performed as described. mIgG: mouse IgG used for ChIP control; AAC02: anti-acetyl lysine antibody used for ChIP; Input: cell lysate prior to ChIP; H2O: Water used as PCR control. The PCR products obtained with GAPDH primers are 166 bp.
Acetylation of proteins can occur as a co-translational or post-translational modification (PTM) (1). Co-translational acetylation occurs at the N-terminal of approximately 85% of mammalian proteins, it is irreversible and is thought to be important in protein stability, localization and synthesis (1). Post-translational acetylation occurs on the epsilon amino group of lysine residues as a reversible and highly dynamic PTM that is known to be a key regulator in multiple cellular events, including chromatin structure, transcription, metabolism, signal transduction and cytoskeletal regulation (2-3). To date over 4,000 proteins have been identified as targets for PTM acetylation which is comparable to phosphorylation in cellular prevelance (3). Antibody AAC01 detects acetyl lysine PTMs.
1 Bogdan P. and Sherman F. 2002. The diversity of acetylated proteins. Genome Biol. 3 (5): reviews 0006.
2 Lundby A. et al. 2012. Proteomic analysis of lysine acetylation sites in rat tissues reveals organ specificity and cellular patterns. Cell Reports 2:419-431.
3 Sadoul K. et al. 2010. The tale of protein lysine acetylation in the cytoplasm. J. Biomed. Biotech. 2011:1-15.
4 Golemis EA et. Al, Protein-Protein Interactions, CSHLP, 2005, p67
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Signal-Seeker™ Acetyl-Lysine Detection Kit (Cat. # BK163)
Signal-Seeker™: BlastR™ Rapid Lysate Prep Kit (Cat. # BLR01)
Signal-Seeker™ Acetylation Affinity Beads (Cat.# AAC04-beads)