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Acetylation Assays Using Acetyl CoA

 

 Overview


3H-acetyl CoA and 14C-acetyl CoA are commonly used radiolabeled substrates for studying in vitro acetylation of histones and other proteins. Acetylation typically occurs at either the N-terminus of a protein or peptide, or on lysine residues within a protein. Alternatively, acetylation can be studied in cells using 3H-sodium acetate. Radioactive substrates for deacetylation assays can be prepared using radiolabeled acetate.

N-terminal acetylation is catalyzed by N-alpha-acetyltransferases (NATs). NATs have been identified as drug metabolizing enyzmes, and have been implicated as biomarkers for certain cancers. The physiological role of N-terminal acetylation is an area of active research.

Histone acetylation and deacetylation on lysine residues plays an important role in epigenetic regulation. Acetylation imparts a negative charge that can neutralize the positive charge on histones, reducing the binding of negatively-charged DNA. This relaxes chromatin structure, making genes accessible to transcription machinery. The acetylation state of histones is balanced by two classes of enzymes: histone acetyltransferases (HATs) and histone deacetylases (HDACs). HATs and HDACs can also acetylate and deacetylate non-histone proteins. HAT and HDAC inhibitors have been studied as potential drugs for treating various cancers.

acetyl_coa_ASK.jpg
Acetylation using labeling acetyl-CoA

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What do I need to run this assay?


Standard in vitro acetylation assays

  • 3H-acetyl CoA or 14C-acetyl CoA (refer to table in next section)
  • Protein substrate
  • Acetyltransferase enzyme
  • Cold acetyl CoA1
  • Gels, buffers, and equipment for polyacrylamide gel electrophoresis (PAGE) analysis2
  • Autoradiography film and developer (or PhosphorImager such as a Cyclone™ for quantitation)2
  • ENLIGHTNING™ autoradiography enhancer (Cat. No. 6NE9741)

1Unlabeled acetyl CoA can be used to decrease the specific activity of the stock radiochemical and increase the molar concentration of acetyl CoA in the reaction.
2As an alternative to gel electrophoresis, the reaction can be separated by washing over Whatman P81 paper and measured on a liquid scintillation counter after the addition of liquid scintillation cocktail.

 

High-throughput FlashPlate® in vitro protein acetyltransferase assay

Reagents available from PerkinElmer:

  • 3H-acetyl CoA or 14C-acetyl CoA (refer to table in next section)
  • Basic FlashPlates or Streptavidin-coated FlashPlates1 (for substrate capture)
  • TopSeal-A™ (Cat. No. 6050185)

1As an alternative to FlashPlates, SPA streptavidin-coated beads are also available


Reagents available from other suppliers:

  • Substrate
  • Acetyltransferase enzyme (HAT)
  • Unlabeled Acetyl CoA

Instrumentation/equipment:

  • High-throughput beta detector (such as a TopCount® counter or MicroBeta® counter)

 

Cell-based acetylation assays

  • 3H- or 14C-sodium acetate (refer to table in next section)
  • Cells
  • Culture media
  • Wash buffer
  • Purification, lysis or extraction reagents
  • Liquid scintillation counter such as a Tri-Carb® counter, vials and cocktail, or SDS-PAGE reagents with film and developer for autoradiography, or SDS-PAGE reagents with ENLIGHTNING™ autoradiography enhancer and PhosphorImager such as a Cyclone™ phosphorimager

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Products and catalog numbers


Radiochemicals

ChemicalRadioisotopeLabelingSpecific activityRad. conc.SolventStorageCat. number
Acetyl-CoA  3Hacetyl-11-10 Ci/mmol0.1 mCi/mLPackaged under nitrogen in 0.01 M sodium acetate pH 5-20°CNET290
14acetyl-1 40-60 mCi/mmol0.2 mCi/mLPackaged under nitrogen in 0.01 M sodium acetate pH 5-20°CNEC313
50-62 mCi/mmol(solid)Freeze-dried solid-20°CNEC313N
Sodium acetate (acetic acid, sodium salt)     3(only one possible labeling position) 75-150 mCi/mmol10 mCi/mLEthanol-20°CNET003
2-5 Ci/mmol10 mCi/mLEthanol-20°CNET003H
14C   (position 1)50-62 mCi/mmol0.2 mCi/mLSteri-packaged, aqueous solution2-8°CNEC084A
(position 1)45-60 mCi/mmol1 mCi/mLEthanol2-8°CNEC084H
(positions 1,2)45-60 mCi/mmol0.1 mCi/mLEthanol-20°CNEC553
(position 2)45-60 mCi/mmol1 mCi/mLEthanol2-8°CNEC085H

How to select a radiochemical from the above table:

  • Chemical
    • Radiolabeled acetyl-CoA is used for biochemical assays (enzymatic asays)
    • Radiolabeled sodium acetate is typically used for cell-based assays
  • Radioisotope: Either 14C- or 3H-radiochemicals can be used in acetylation assays. 14C has higher energy compared to 3H. 14C also has higher efficiency compared to 3H in liquid scintillation counting. Your radioactive license may restrict you to one radioisotope or the other. You will want to consult your radiation safety officer when selecting a radioisotope for your assay.
  • Packaging
    • Radiochemicals that are packaged in ethanol are typically more-stable
    • For cell labeling or in vivo experiments, you may need to evaporate off any ethanol in the stock material prior to use, as ethanol can be toxic to cells.
  • Specific activity: the higher the specific activity, the greater the amount of radioactivity there is per acetyl CoA or acetate molecule. Specific activity is given in units of Curies per millimole of acetyl CoA or sodium acetate, or milliCuries per millimole of acetyl CoA or sodium acetate. The theoretical maximum specific activity of tritium is ~29 Ci/mmol (Curies per millimole of tritium). 3H products with a specific activity that approaches this value have a greater proportion of molecules labeled with 3H in the stock vial. Note that NET003H has a higher specific activity than NET003 (Ci/mmol vs. mCi/mmol). The theoretical maximum specific activity of 14C is ~62 mCi/mmol (milliCuries per millimole of carbon). 14C products with a specific activity that approaches this value have a greater proportion of molecules labeled with 14C in the stock vial. Specific activity can be decreased by adding cold (unlabeled) acetyl CoA or sodium acetate.

 

Autoradiography enhancers for film or phosphorimaging

  • EN3HANCE® Autoradiography Enhancer (Cat. No. 6NE9701): use on gels for best band resolution
  • ENLIGHTNING™ Rapid Autoradiography Enhancer (Cat. No. 6NE9741): safer alternative to 6NE9701. Use on gels.

 

Scintillation cocktails

  • Ultima Gold™ F (Cat. No. 6013179): can be used as scintillation cocktail for dry filters including glass fiber, cellulose nitrate, cellulose acetate, mixed cellulose esters, and normal paper filters
  • Ultima Gold (Cat. No. 6013326): multipurpose liquid scintillation cocktail for aqueous and non-aqueous samples


Scintillation vials

 

FlashPlates

  • Basic FlashPlate 96 (Cat. No. SMP200 - for direct-coating of substrate to plate)
  • Basic FlashPlate 384 (Cat. No. SMP400 - for direct-coating of substrate to plate)
  • Streptavidin FlashPlate 96 (Cat. No. SMP103 - for indirect capture of biotinylated substrate to plate)
  • Streptavidin FlashPlate 384 (Cat. No. SMP410 - for indirect capture of biotinylated substrate to plate)


Detection instruments

  • Tri-Carb® liquid scintillation counter
  • Cyclone™ phosphorimager
  • TopCount® high-throughput detector for plates
  • MicroBeta® high-throughput detector for plates (and for some models, 4 mL vials and Eppendorf tubes)

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Protocol-in-brief


acetyl_workflow_ASK.jpg
Acetylation workflow

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Guides


  • Link to the FlashPlates knowledge base page

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Citations


Standardin vitroacetylation assays

  1. Pradeepa, M.M. et al. Acetylation of Transition Protein 2 (TP2) by KAT3B (p300) Alters Its DNA Condensation Property and Interaction with Putative Histone Chaperone NPM3. Journal of Biological Chemistry 284:, 29956 -29967 (2009). Link
  2. Stimson, L. et al. Isothiazolones as inhibitors of PCAF and p300 histone acetyltransferase activity. Mol. Cancer Ther 4, 1521-1532 (2005). Link
  3. Lau, O.D. et al. p300/CBP-associated Factor Histone Acetyltransferase Processing of a Peptide Substrate: KINETIC ANALYSIS OF THE CATALYTIC MECHANISM. Journal of Biological Chemistry 275, 21953-21959 (2000). Link
  4. Herrera, J.E., Bergel, M., Yang, X., Nakatani, Y. & Bustin, M. The Histone Acetyltransferase Activity of Human GCN5 and PCAF Is Stabilized by Coenzymes. Journal of Biological Chemistry 272, 27253-27258 (1997). Link


FlashPlatein vitroacetylation assays

  1. Stimson, L. et al. Isothiazolones as inhibitors of PCAF and p300 histone acetyltransferase activity. Mol. Cancer Ther 4, 1521-1532 (2005). Link
  2. Wynne Aherne, G., Rowlands, M.G., Stimson, L. & Workman, P. Assays for the identification and evaluation of histone acetyltransferase inhibitors. Methods 26, 245-253 (2002). Link
  3. Turlais, F. et al. High-throughput screening for identification of small molecule inhibitors of histone acetyltransferases using scintillating microplates (FlashPlate). Anal. Biochem 298, 62-68 (2001). Link


Cell-based acetylation assays

  1. Guo, C., Mi, J., Brautigan, D.L. & Larner, J.M. ATM regulates ionizing radiation-induced disruption of HDAC1:PP1:Rb complexes. Cell. Signal 19, 504-510 (2007). Link
  2. Kraker, A.J. et al. Modulation of Histone Acetylation by [4-(Acetylamino)-N-(2-Amino-phenyl) Benzamide] in HCT-8 Colon Carcinoma. Molecular Cancer Therapeutics 2, 401-408 (2003). Link
  3. Verdin, E. et al. Measurement of Mammalian Histone Deacetylase Activity. Chromatin and Chromatin Remodeling Enzymes, Part C Volume 377, 180-196 (2003). Link

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Tips


  • Published literature suggests that histones can intrinsically attach to the surface of FlashPlates. Therefore, if you are running a histone acetylation assay on FlashPlates, you have the option of mixing the substrate, 3H-acetyl CoA, and enzyme all at once, incubating, then measuring. No pre-coating of the FlashPlate is required.
  • Acetyl CoA can be unstable. Very specific conditions are required to prevent hydrolysis. If you are preparing unlabeled ("cold") Acetyl-CoA for your assay, it is usually best to prepare this fresh from powder.

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Other PerkinElmer protein acetylation assay technologies


Non-radioactive AlphaScreen® or AlphaLISA® no-wash acetylation assays

LANCE® TR-FRET acetylation assays

LabChip® EZ Reader microfluidic MSA assays

DELFIA® fluorescent histone acetylation and other post-translational modification citations

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Custom services at PerkinElmer


PerkinElmer offers custom radiochemicals and custom assay development services. If you are interested in custom services, please contact our custom teams:

ON>POINT® Custom Services

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