Details Host/IsotypeMouse / IgG2bClone7B5A1Species ReactivityAll speciesValidation DataAc-K Antibody White PaperIF: Ac-K MitochondriaAAC02, 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; in particular, it can be used to visualize acetylated mitochondrial proteins by IF. Validated Applications Western Blot using Acetyl-Lysine Antibody (AAC02) 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.  AAC02 was used at a 1:500 dilution following the recommended western blot protocol.To see the full Western blot comparison, see the Optimized Protocols or the product datasheet.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 μl of AAC02 was used per IP reaction. Western blots of immunoprecipitated proteins were developed using AAC03-HRP at 1:3000 dilution.To see the full Immunoprecipitation comparison, see the Optimized Protocols or the product datasheet.Immunofluorescence using Acetyl-Lysine Antibody  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 AAC02 binding as an indicator of AAC02 specificity for acetyl-lysine modifications.To see the full Immunofluorescence protocol, see the Optimized Protocols or the product datasheet.AAC02 detects mitochondrial acetylation by IF Fig 4: Swiss 3T3 cells, either untreated or treated with TSA (1 μM for 6h), were stained as described in the method. (a and d): Mitochondria were visualized with MitoTracker orange (Thermo Fisher). (b and e): Acetylated proteins were visualized in green fluorescence. (c and f): Merged image of mitochondrial and acetylation signals. Actin fibers and nuclei were visualized in purple with Rhodamine Phalloidin and blue with DAPI respectively. Note: AAC02 provides broad, pan acetyl-lysine detection including acetylated mitochondrial , nuclear, and cytoplasmic proteins. To see the full Immunofluorescence protocol, see the Optimized Protocols or the product datasheet.ChIP using Acetyl-Lysine Antibody Fig 5: Utilization of AAC02 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.  To see the recommended ChIP protocol, see the Optimized Protocols or the product datasheetProtein Acetylation BackgroundAcetylation 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.  References1 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, p67For more information contact:  signalseeker@cytoskeleton.comAssociated Products: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)AboutFor product Datasheets, MSDSs, and COAs please click on the PDF links below. Sample Size Datasheet (Cat. AAC02-s):  Certificate of Analysis:  available upon request CitationsFor the most recent publications citing this and other Signal-Seeker™ products, see our Signal-Seeker™ Validation Data Page click hereFaqsVisit our Signal-Seeker™ Tech Tips and FAQs page for technical tips and frequently asked questions regarding this and other Signal-Seeker™ products click here If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com