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Epigenetics & Chromatin

Nucleosome Preparation Kit

easily isolate mononucleosomes and oligonucleosomes

Active Motif’s Nucleosome Preparation Kit is designed to prepare mono- and oligonucleosomes starting from cells. Nucleosomes comprise the smallest subunit of chromatin and consist of 147 bp of DNA wrapped around an octamer of core histone proteins (H2A, H2B, H3 and H4). Each nucleosome is separated by a linker of DNA. The Nucleosome Preparation Kit uses an enzymatic cocktail to digest the linker DNA. By adjusting the digestion time, it is possible to control the amount of mononucleosomes or oligonucleosomes extracted. Because isolated nucleosomes are more physiologically relevant than histones alone or synthetic peptides, they are ideally suited for use as substrates in the analysis of histone post-translational modifications, enzyme kinetics or inhibitor screening studies.

Nucleosome Preparation Kit preserves intact nucleosomes.
Gel shift showing preservation of intact nucleosomes.

Nucleosome Advantages:

  • More physiologically relevant substrates than synthetic peptides
  • Useful for the analysis of histone post-translational modifications, enzyme kinetics or inhibitor screening studies
  • Easily adjust the levels of mononucleosomes or oligonucleosomes isolated by adjusting digestion times
 
Name Format Cat No. Price  
Nucleosome Preparation Kit 20 rxns 53504 $255 Buy Now

Easily prepare mononucleosomes or oligonucleosomes from your samples

The ability to regulate the amount of mononucleosomes or oligonucleosomes isolated with the Nucleosome Preparation Kit based on digestion time with the Enzymatic Shearing Cocktail is shown in Figure 1. A comparison of isolated nucleosomes with purified nucleosome DNA in Figure 2 illustrates the gel shift observed in the molecular weight of the nucleosome sample, thereby confirming the nucleosome is intact with DNA wrapped around the histone proteins.

Efficiency of nucleosome digestion into mononucleosomes and oligonucleosomes
Figure 1: DNA gel analysis of nucleosome digestion times.

Nucleosomes were prepared from HeLa cells and digested with the Enzymatic Shearing Cocktail for 5, 10 and 15 minutes. Following digestion, the reactions were stopped with the addition of ice-cold EDTA. The nucleosomes were then subjected to DNA clean up to assess the digestion efficiency. Samples were separated by electrophoresis through a 1.5 % agarose gel

Lane 1: 100 to 1000 bp DNA ladder.
Lane 2: HeLa genomic DNA.
Lane 3: HeLa DNA digested for 5 minutes.
Lane 4: HeLa DNA digested for 10 minutes.
Lane 5: HeLa DNA digested for 15 minutes.

 

Comparison of intact nucleosomes and purified DNA shows gel shift for assembled nucleosomes.
Figure 2: Gel shift observed with intact nucleosomes as compared to purified nucleosome DNA.

Nucleosomes were prepared from HeLa cells according to the instructions in the Nucleosome Preparation Kit. A 50 l aliquot of extracted nucleosomes was then subjected to DNA clean up to assess the digestion efficiency. Both intact nucleosomes and purified DNA samples were analyzed by TapeStation. The results show an increase in molecular weight banding for the intact nucleosomes as compared to the purified DNA samples. This is predicted since an intact nucleosome complex contains both the DNA and histone proteins.

Lane A: 25 to 1500 bp DNA ladder.
Lane B1: Intact nucleosome samples.
Lane C1: Purified nucleosome DNA.

Contents & Storage

Please note that the Nucleosome Preparation Kit is shipped on dry ice and contains reagents with multiple storage temperatures inside. Please store each component at the temperature indicated below. All reagents are guaranteed stable for 6 months from date of receipt when stored properly. This kit includes enough reagents for 20 preparations from 10 cm plates and 5 sample optimizations.

  • 10X PBS; Store at 4°C to -20°C
  • 100 mM PMSF; Store at -20°C
  • Protease Inhibitor Cocktail (PIC); Store at -20°C
  • Lysis Buffer; Store at -20°C
  • Digestion Buffer; Store at 4°C to -20°C
  • Enzymatic Shearing Cocktail; Store at -20°C
  • 0.5 M EDTA; Store at 4°C to -20°C
  • RNase A (10 µg/µl); Store at -20°C
  • Proteinase K (10 µg/µl); Store at -20°C
  • 5 M NaCl; Store at -20°C
  • 3 M Sodium Acetate; Store at RT