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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.
Advantages
- Convenient spin column format
- Preserves modifications on histones
- Purify histones from as few as 8x105 cells
- Useful for purifying histones from budding yeast
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. It is even possible to purify enriched histone fractions from budding yeast. 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, rat brain tissue, budding yeast) 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.
- Yeast: 0.5 mg total core histones from 8 x 107 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.
