Chromatin Assembly Kit
Chromatin-Assembly-Kit-AM13
Synonyms
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Overview
The Chromatin Assembly Kit enables you to study your DNA sequence of interest in a native chromatin environment. The simple, easy-to-follow protocol generates assembled chromatin in hours with few manipulations, providing you with material that is ideal for downstream applications such as in vitro ChIP, transcription and histone modification assays.
To study specific histone modifications within a chromatin context, Active Motif's Recombinant Histones with site and degree-specific modifications can be used as substrates for chromatin assembly.
Description
The Chromatin Assembly Kit includes native HeLa core histones and recombinant chaperone and assembly proteins for the in vitro assembly of nucleosomes in a natural, ordered array on either circular or linear input DNA. The kit also includes reagents to confirm the ordered array of nucleosomes on the DNA with a simple and quick partial enzymatic digestion. The partial digestion clearly shows the ordered spacing of nucleosomes on the DNA (Figure 1).
Figure 1: Digestion of chromatin assembled from supercoiled circular and linear DNA. One hundred µl reactions were performed on 1 µg of either linear or circular DNA using the Chromatin Assembly Kit protocol. Following the chromatin assembly incubation, each reaction was divided in two, and partially digested with 0.5 µl Enzymatic Shearing Cocktail for 2 and 4 minutes, respectively. Samples were subsequently deproteinated with Proteinase K, phenol/chloroform extracted and analyzed by agarose gel electrophoresis.
Lane 1: 100 bp DNA ladder.
Lane 2: Circular DNA digested with Enzymatic Shearing Cocktail for 2 minutes.
Lane 3: Circular DNA digested with Enzymatic Shearing Cocktail for 4 minutes.
Lane 4: Linear DNA digested with Enzymatic Shearing Cocktail for 2 minutes.
Lane 5: Linear DNA digested with Enzymatic Shearing Cocktail for 4 minutes.
Chromatin Assembly Kit advantages
- Generate chromatin from linear or supercoiled DNA
- ATP-dependent method results in an extended array of regularly spaced nucleosomes
- Positive control supercoiled DNA provided to ensure success
- Easy protocol, simply add components and incubate
- Excellent substrate for gene regulation experiments
Applications
The chromatin generated with the Chromatin Assembly Kit can be used in many applications, such as investigation of factors involved in transcription activation by in vitro transcription assays. In addition, you can also determine which factors are involved with histone acetylation by performing in vitro histone acetylation (HAT) assays. You can also perform an in vitro ChIP assay of the assembled chromatin to verify transcription factor binding.
Contents
Contents & Storage
Recombinant hNAP-1 protein, purified HeLa Core Histones, recombinant ACF complex, high and low salt buffers, 10X ATP regeneration system, Creatine Kinase, control supercoiled DNA, Enzymatic Shearing Cocktail, Proteinase K and Enzymatic Stop solution, glycogen, 5X Orange G Dye, and siliconized microcentrifuge tubes. Storage conditions range from room temperature to -80°C, see the manual for specific details. Contents are guaranteed for six months from delivery when stored properly.
The purified HeLa Core Histones are also sold separately.