Histone Purification Mini Kit

Synonyms

histone, histones, purification, isolation, H2A, H2B, H3, H4, H2, core histones, purify, post-translational modifications, acetylation, methylation, phosphorylation, H2A/H2B, H3/H4, spin column, purification column, chromatin

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Overview

Histone Purification Mini Kit Overview

Active Motif's Histone Purification Mini Kit enables you to purify core histones (H2A, H2B, H3 and H4) as one total population while preserving their post-translational modifications such as acetylation, methylation, and phosphorylation states. This purification method is an improvement over acid precipitation methods and utilizes a convenient spin column and proprietary buffer system to purify the core histones from cells and tissue samples. Histones isolated by this method are suitable substrates for downstream assays and in vitro chromatin assembly.

Histone Purification Mini Kit Advantages:

• Only commercial kits available for histone purification
• Convenient spin column format
• Preserves modifications on histones
• Purify histones from as few as 8x10⁵ cells
• Works with cells or tissue samples

How does it work?

Unlike histone purification by acid precipitation, our method utilizes a unique purification column and a series of proprietary elution buffers to isolate pure histone fractions. The column has a high binding capacity for histones, so core histones can be isolated from small cell culture samples or from grams of tissue. Purification of histones using the Histone Purification Mini Kit also preserves key post-translational modifications, such as acetylation, methylation and phosphorylation (Figure 1).

Figure 1: Analysis of histone fractions by SDS-PAGE and Western blot
Histones were purified from a variety of sources (unstimulated HeLa cells, sodium butyrate-treated HeLa cells, Paclitaxel-treated HeLa cells, and rat brain tissue) using the Histone Purification Mini Kit. The first and second elutions (labeled 1st and 2nd) from each sample were analyzed on a 16% Tris-glycine SDS-PAGE gel (top panel) and by Western blot (bottom panel). Antibodies recognizing acetyl-Histone H3 (Ac-H3, Catalog No. 39139, 1:500 dilution), phospho-Ser28 Histone H3 (Phos-H3, Catalog No. 39098, 1:1,000 dilution), and trimethyl-Lys4 Histone H3 (Me-H3, Catalog No. 39159, 1:1,000 dilution).

Efficient histone yield from small numbers of cells

The Histone Purification Mini Kit provides reagents for 20 histone purifications from as little as a 8x10⁵ cells up to grams of tissue due to the robust histone binding capacity of the purification column. The kit method is simple:  first, an extract is made and applied to the purification column, then histones are eluted, enabling the purification of core histones. Histones may be quantified by an OD reading or quantitated empirically on a gel by comparison with histone standards.

Yield of Histones

The following yields are approximate. Results may vary according to cell or tissue type. It is possible to obtain a decent yield (10 µg) of histones from as few as 8 x 10⁵ mammalian cells (Figure 2).

• Adherent Cells: 0.1 mg total core histones from 8 x 10⁶ cells (one 150 mm plate)
• Suspension Cells: 0.1 mg total core histones from 8 x 10⁶ cells
• Tissue: 1 mg histone per gram of tissue*

*The theoretical binding limit for these columns is 1 mg, but this has not been confirmed empirically. Loading more than the theoretical limit is not recommended.

Figure 2:  Analysis of histone yield using varying numbers of HeLa cells
Histones were extracted from different numbers of HeLa cells using the Histone Purification Mini Kit and loaded onto a 16% SDS-PAGE gel. Purified HeLa core histones were loaded as a standard in parallel.

N.B., on this particular gel system, H2A and H2B exhibit coincident migration.
Lane 1: 15 µl of a 100 µl elution from histones purified from a single 150 mm tissue culture plate of HeLa cells at 100% confluency (8 x 10⁶ cells). Lane 2: 15 µl of a 50 µl elution from histones purified from one-half of the number of cells from a 150 mm tissue culture plate of HeLa cells at 100% confluency (4 x 10⁷ cells). Lane 3: 15 µl of a 50 µl elution from histones purified from one-fifth the number of cells from a 150 mm tissue culture plate of HeLa cells at 100% confluency (1.6 x 10⁷ cells). Lane 4: 15 µl of a 50 µl elution from histones purified from one-tenth the number of cells from a 150 mm tissue culture plate of HeLa cells at 100% confluency (8 x 10⁵ cells). Lane 5: 2.5 µg of purified HeLa core histones. Lane 6: 5 µg of purified HeLa core histones.

Contents

Histone Purification Mini Kit Contents

Inhibitors are NOT included in the Histone Purification Mini kit and are generally not required to preserve histone post-translational modifications using this kit. If inhibitors are desired, they must be supplied by the user and can be added to the extraction and neutralization buffers during isolation. Additional Extraction Buffer and 5X Neutralization buffer may be purchased separately.

Please note that the Histone Purification Mini Kit is shipped at room temperature 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. Each Histone Purification Mini Kit supplies sufficient reagents to perform 20 purifications. This kit includes the following components:

• Extraction Buffer; Store at 4°C
• 5X Neutralization Buffer; Store at 4°C
• Equilibration Buffer; Store at 4°C
• Histone Wash Buffer; Store at 4°C
• Histone Elution Buffer; Store at 4°C
• Purification spin columns; Store at RT
• Collection tubes; Store at RT

Histone Purification Mini Kit spin column in a collection tube.

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