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Color-de-Lys® HDAC colorimetric activity assay kit

Simplify HDAC activity analysis with the fastest colorimetric assay on the market
 
BML-AK501-0001 96 wells 374.00 USD
 
  • Simple two-step protocol with < 1 hour time to answer
  • Colorimetric readout at 405 nm compatible with most plate readers
  • Resistant to detergent interference common to antibody-based assays
  • Eliminates need for radioactivity, extractions, and/or chromatography
  • Suitable for high throughput analysis
     
Useful for assaying lysates, immunoprecipitates or inhibitor screening using the nuclear extract provided.- Includes HeLa nuclear extract, a rich source of HDACs 1 & 2 for use as a positive control or as a source of HDAC activity for screening.
Compatible with class I & IIb HDAC and sirtuins (with addition of NAD+).
The Color de Lys® system (Colorimetric Histone de Acetylase Lysyl Substrate/Developer) is a sensitive and convenient alternative to protocols utilizing radiolabeled, acetylated histones or peptide/HPLC methods for the assay of histone deacetylases. The assay procedure has two steps. First, the Color de Lys® substrate which comprises an acetylated lysine side chain, is incubated with a sample containing HDAC activity (HeLa nuclear or other extract, purified enzyme, bead bound immunocomplex, etc.). Deacetylation of the substrate sensitizes the substrate so that, in the second step, mixing with the Color de Lys® developer causes an increase in yellow color intensity, and absorption at 405 nm.
Note: The Color de Lys® substrate is efficiently deacetylated by HDAC1 and HDAC2, the major contributors to HDAC activity in HeLa nuclear extracts. It is however, a poor substrate for HDAC3 and recombinant human HDAC8 (BIOMOL International, unpublished results). The activities of other HDAC isotypes with the Color de Lys® substrate have yet to be investigated. HDAC3 and HDAC8 do deacetylate the Fluor de Lys® substrate, which is the basis for the HDAC fluorometric activity assay kit (Prod. No. BML-AK500).
BML-AK501 HDAC graph
Our Color-de-Lys HDAC colorimetric assay is less sensitive to detergents than competitive antibody-based assays. HeLa nuclear extract (8.3µg) was added to the substrate and buffer recommended by the manufacturer in the presence or absence of 0.1% Triton X-100. After 60 minutes at 37°C, the reaction was stopped and processed as recommended by the manufacturers. Triton X-100 showed little or no effect on the Color-de-Lys reaction, but caused an apparent 70% inhibition of the antibody-based assay.
BML-AK501 schematic
Reaction Scheme of the HDAC Colorimetric Activity Assay (patent pending). Deacetylation of the substrate sensitizes it to the developer, which causes an increase in yellow intensity and absorption at 405 nm.
BML-AK501 std curve
Please mouse over
BML-AK501 HDAC graph BML-AK501 schematic BML-AK501 std curve

Product Specification

Alternative Name:Histone deacetylase colorimetric assay kit
 
Use/Stability:Store all components except the microtiter plate at -70°C for the highest stability. The HeLa Nuclear Extract must be handled with particular care in order to retain maximum enzymatic activity. Defrost it quickly in a RT water bath or by rubbing between fingers, then immediately store on an ice bath. The remaining unused extract should be refrozen quickly, by placing at -70°C. If possible, snap freeze in liquid nitrogen or a dry ice/ethanol bath. To minimize the number of freeze/thaw cycles, aliquot the extract into separate tubes and store at -70°C. The Color de Lys™ Substrate, when diluted in Assay Buffer, may precipitate after freezing and thawing. It is best, therefore, to dilute only the amount needed to perform the assays of that day.
 
Long Term Storage:-80°C
 
Kit/Set Contains:

Nuclear Extracts from HeLa Cells (human cervical cancer cell line) (Prod. No. BML-KI137) (500 µl) Storage: avoid freeze/thaw cycles!
Color de Lys® Substrate (Prod. No. BML-KI138) (50 µl; 50mM in DMSO) Storage: -70°C
Color de Lys® Developer Concentrate (20x) (Prod. No. BML-KI139) (100 µl; 20x stock solution, dilute in HDAC assay buffer before use) Storage: -70°C
Trichostatin A (HDAC Inhibitor) (Prod. No. BML-GR309-9090) (100 µl; 0.2mM in DMSO) Storage: -70°C
Color de Lys® Deacetylated Standard (Prod. No. BML-KI141) (30 µl; 10mM in DMSO) Storage: -70°C
HDAC Assay Buffer (50mM TRIS-HCl, pH 8.0, 137mM sodium chloride, 2.7 mM potassium chloride, 1mM magnesium chloride) (Prod. No. BML-KI143) (20 ml) Storage: -70°C
1/2 volume microplate (Prod. No. BML-KI101) Storage: Room temperature

 

Product Literature References

Phosphodiesterase-4 inhibition improves corticosteroid insensitivity in pulmonary endothelial cells under oxidative stress: J.L. Ortiz, et al.; Allergy 68, 64 (2013), Abstract;
Family with sequence similarity 60A (FAM60A) protein is a cell cycle-fluctuating regulator of the SIN3-HDAC1 histone deacetylase complex: I.M. Munoz, et al.; J. Biol. Chem. 287, 32346 (2012), Application(s): HDAC activity in nuclear extracts from HEK293 cells, Abstract; Full Text
Glucose depletion activates mmu-miR-466h-5p expression through oxidative stress and inhibition of histone deacetylation: A. Druz, et al.; Nucleic Acids Res. 40, 7291 (2012), Application(s): HDAC activity in nuclear extracts from mouse cell lines, Abstract; Full Text
Long-acting β2 agonists and corticosteroids restore the reduction of histone deacetylase activity and inhibit H2O2-induced mediator release from alveolar macrophages: D.W. Perng, et al.; Pulm. Pharmacol. Ther. 25, 312 (2012), Application(s): HDAC activity in nuclear extracts from alveolar macrophages, Abstract;
Sulforaphane causes a major epigenetic repression of myostatin in porcine satellite cells: H. Fan, et al.; Epigenetics 7, 1379 (2012), Abstract;
Histone deacetylase inhibitor activity in royal jelly might facilitate caste switching in bees: A. Spannhoff, et al.; EMBO Rep. 12, 238 (2011), Application(s): Inhibition study using isolated histones, Abstract; Full Text
SIRT1 is a redox-sensitive deacetylase that is post-translationally modified by oxidants and carbonyl stress: S. Caito, et al.; FASEB J. 24, 3145 (2010), Application(s): Activity of immunoprecipitated SIRT1, Abstract; Full Text

General Literature References

Isolation and characterization of mammalian HDAC10, a novel histone deacetylase: H. Y. Kao, et al; J Biol. Chem. 277, 187 (2002), Abstract; Full Text
Acetylation and chromosomal functions: W.L. Cheung, et al.; Curr. Opin. Cell Biol. 12, 326 (2000), Abstract;
Cloning and characterization of a novel human class I histone deacetylase that functions as a transcription repressor: E. Hu et al.; J. Biol. Chem. 275, 15254 (2000), Abstract;
Histone deacetylases: silencers for hire: H.H. Ng et al.; Trends Biochem. Sci. 25, 121 (2000), Abstract;
The language of covalent histone modifications: B.D. Strahl et al.; Nature 403, 41 (2000), Abstract;
Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase: S. Imai et al.; Nature 403, 795 (2000), Abstract;
A new family of human histone deacetylases related to Saccharomyces cerevisiae HDA1p: W. Fischle et al.; J. Biol. Chem. 274, 11713 (1999), Abstract;

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