Acetylation of proteins can occur as a co-translational or post-translational modification (PTM) (1). Co-translational acetylation occurs at the N-terminus of approximately 85% of mammalian proteins, 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 prevalence (3). By using the protocols below anti-acetyl lysine can be used to detect and measure acetylated proteins involved in signal transduction pathways. Acetyl lysine protocols are useful to detect and measure tubulin, histone, histone1, PVDF membrane, PVDF probing, histone2, histone 3, histone4a, histone 4b. acetylated protein analysis, WB , IP, ChIP, IF, acetylated protein, p53, sirtuin, hdac1
Product Citations for Anti-acetyl lysine monoclonal antibody (Cat. AAC01) (since 2015 introduction):
LaBarge et al. 2015. p300 is not required for metabolic adaptation to endurance exercise training. The FASEB Journal article doi: 10.1096/fj.15-281741.
, hdac2, hdac3, hdac3, hdac4, hdac5, hdac6, hdac, histone de-acetylase, tat, tubulin acetyl transferase, histone acetyl transferase, acetyl transferase.
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.
Use as indicated below at 1:500-1:1000 dilution, sufficient for 100-200 ml of working strength Ab.
NOTE: This Ab has some cross reactivity with the dyes used for pre-stained molecular weight markers. This does not affect protein specificity for acetyl groups (see Fig. 1B -1C). Also, some molecular weight marker proteins are acetylated, particularly commonly used metabolic enzymes such as glutamic dehydrogenase (m.wt. approx. 55kDa).
Figure 1: Western blot applications
Legend: AAC01 was used at a 1:500 dilution following the recommended Western blot protocol (see below). Figure 1A: murine tissue extract, 30 g liver extract. Figure 1B: 20 g of A431 cell lysate treated with TSA (+) or untreated (-). Strongly enhanced bands at 55 and 14-16 kDa in TSA-treated lysate correspond to acetylated tubulin and histone proteins, respectively. Figure 1C: 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. AAC01 recognizes 0.005 ng of chemi-cally acetylated BSA. For comparison ImmuneChem anti-acetyl lysine antibody Cat# ICP0380 was able to detect 0.05 ng of acetylated BSA when used as instructed by manufacturer. Arrow-head indicates acetylated BSA, higher molecular weight bands are acetylated BSA oligomers.
Use as indicated at 20 µl per IP reaction (1.5 mg total lysate per IP), sufficient for approximately 10 IP assays.
Figure 2: IP of histone proteins from TSA-treated A431 cells
Legend: A431 cells were either treated (+) or untreated (-) with TSA (0.6 mM for 6 h). Cell lysates were prepared in a modified RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% IGEPAL, 0.1% SDS, 0.5% Na deoxycholate) and 1.5 mg of lysate per reaction was used for IP of acetylated histones. Each IP was performed according to the manufacturer’s instructions. IP reactions for AAC01 are shown in duplicate. Western blots of immunoprecipitated proteins were developed using AAC01 at 1:500 dilution. Input signal is from 15 mg of TSA treated (+) or untreated (-) A431 lysates. Ability of AAC01 to IP histones was compared to other commercially available antibodies, ThermoFisher (Pierce) Cat. # MA1-2021, ImmuneChem Cat. # ICP0388 and Cell Signaling Cat. # 9441. IPs were carried out according to the manufacturer’s instructions.
Use as indicated below at 1:200 dilution, sufficient for 40 ml of working strength Ab, approx. 400 IF slides.
IF Method (TSA treatment of A431 cells)
Figure 3: IF of A431cells untreated and treated with TSA
Legend: Human epidermoid carcinoma A431 cells, untreated (left) or treated (right) with TSA (5 µM for 16 h), were stained as described in the method. Acetylated cytoplasmic and nuclear proteins were visualized in green fluorescence. Note that in contrast with the untreated control, acetylated microtubule network is clearly visible in TSA-treated sample. The fluorescent nuclear intensities indicate the high abundance of acetylated proteins in the nucleus.
Use as indicated below at 1:100 dilution, sufficient for 200 ChIP assays of 100 µl volume.
Figure 4: ChIP analysis with AAC01 antibody
Legend: Chromatin was prepared from A431 cells, either untreated or TSA-treated (5 mM, 4 hrs). ChIP was performed according to method see below. Ctr IgG: mouse IgG used for ChIP, AAC01: anti-acetyl antibody used for ChIP, Input: cell lysate prior to ChIP. PCR analysis was carried out using primers for the promoter region of the housekeeping gene GAPDH.