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Epigenetics & Chromatin

Histone Purification Microplate Kit

histone purification system for high throughput applications

Active Motif's Histone Purification Microplate Kit enables purification of core histones (H2A, H2B, H3 and H4) using very small amounts (as little as 0.9 x 105 cells) of starting material, and its microplate spin column format enables elution into a 96-well plate to easily transition into downstream high-throughput applications, including our Histone Modification ELISAs and Luminex® Histone H3 PTM Multiplex Assays to screen for changes in histone modifications.

PBMC ChIP on CD8+ T Cells
Achieve better yields with Histone Microplate Purification than with crude extraction.

(Click image to enlarge)

The Histone Purification Microplate Kit method utilizes the identical proprietary buffer system used in our Histone Purification Kit and our Histone Purification Mini Kit to purify core histones from cells and tissue samples. This purification method is superior to acid precipitation methods because it preserves post-translational histone modifications and greatly improves the purity and yield of your isolated histones to improve the reliability and consistency of your sample preparation. Histones isolated by this method are excellent substrates for use in downstream high-throughput screening assays, Western blot or mass spectrometry.

To learn more about the Histone Purification Microplate Kit, click on the Method, Data, or Contents tabs below. To view a manual or other related documents, click on the Documents tab below.

Advantages

  • Only commercial kits available for histone purification
  • Higher quality sample prep than crude acid extraction
  • 96-well format with modular stripwell design for flexibility in throughput
  • Preserves modifications on histones
  • Purify histones from as few as 0.9x105 cells
  • Works with cell or tissue samples
 
Name Format Cat No. Price  
Histone Purification Microplate Kit 96 rxns 40027 $810 Buy Now

How does it work?

The Histone Purification Microplate Kit is designed to provide researchers with a rapid method for purification of core histones to streamline into downstream high throughput screening applications. The method improves yield, purity and concentrations above what is achievable with crude acid extraction, providing the highest quality substrate for use in biopharmaceutical and screening assays.

The kit supplies enough reagents to perform up to 96 histone purifications starting with as little as a 0.9x105 cells. The method utilizes column filtration to simplify and speed up the processing of samples. The columns are provided in a 96-well plate spin column format that features a modular 8-strip unit design to allow adaptation to both low and high throughput processing.

Histone Purification Microplate

The Histone Purification Microplate Kit utilizes a series of proprietary elution buffers that are identical to those used in our Histone Purification Kit and our Histone Purification Mini Kit to purify core histones from cells and tissue samples. Purification of histones using this method eliminates contaminating acid-insoluble cellular proteins that are left behind with standard acid extraction protocols. Removal of these impurities also prevents any residual alterations to the histone modifications so that key post-translational modifications on histone tails are preserved, providing you with the highest quality histone samples for use in your downstream analysis.

The kit method is simple. First, make an extract from your cell or tissue samples. Then, apply the extract to the purification, elute histones and quantititate. Core histones (H2A, H2B, H3 and H4) are eluted as one highly concentrated total population.

Quantification of histone proteins

Quantification of histone proteins from small cell numbers is not possible using traditional methods for protein quantification, such as Bradford or absorbance at 230 nm, as the yield will fall below the detectable range for these methods. Instead, we suggest using Active Motif’s Histone H3 Total ELISA Kit to quantify the amount of histone H3 recovered. ELISAs for specific histone modifications are also available to quantify the amount of a particular histone PTM within the sample. Alternatively, Active Motif’s Histone H3 PTM Multiplex Assay can be used in combination with Total Histone H3 antibody-conjugated bead set to screen histone H3 levels.

  • Histone H3 Total ELISA Kit (Cat. No. 53110) – we suggest using a starting volume of 2 µl per well of purified histones extracted from 1 x 105 cells in the ELISA.
  • Histone Modification ELISAs – we suggest using a starting volume of 20 µl per well of purified histones extracted from 1 x 105 cells in the ELISA.
  • Histone H3 PTM Multiplex Assay (Cat. No. 33115) – we suggest using a starting volume of 10 µl per well of purified histones extracted from 1 x 105 cells in the assay.

Achieve the Highest Quality Samples for Your High Throughput Applications

The Histone Purification Microplate Kit provides a fast, reliable and high throughput method for for obtaining high quality histone samples for use in downstream applications. The proprietary method enables purification of core histones at significantly higher yields and purity than what is achievable by crude extraction.

Purity of the core histones isolated with the Histone Purification Microplate Kit
Figure 1: SDS-PAGE comparison of the purity of histone fractions isolated by crude acid extraction or purified using the Histone Purification Microplate Kit.

Histones were extracted from 2 x 105 HeLa cells using either crude acid extraction conditions (Lanes 1-3) or the Histone Purification Microplate Kit (Lanes 4-6). Nine µl of each biological replicate were loaded and run on a 4-20% SDS gradient gel, then stained with Ponceau Stain. The crude acid extracted material shows contamination of other acid soluble proteins in the elution, while the Histone Purification Microplate Kit yields a pure fraction of core histones.


Histone Purification Microplate Method Preserves Post-translational Modifications

Histone Purification Microplate Kit preserves post-translational modifications
Figure 2: Histone Purification Microplate Kit preserves post-translational modifications.

Active Motif's Histone Purification Microplate Kit preserves phospho, acetyl and methyl post-translational modifications (PTMs) on histones. The Western blot data shown above of paclitaxel-treated HeLa (lane 1), sodium butyrate-treated HeLa (lane 2), and unstimulated HeLa (lane 3) purified from 0.75 x 106 cells demonstrates that the PTMs are intact following purification of histones.


Achieve Higher Yields than with Crude Acid Extraction

The Histone Purification Microplate Method enables you to extract significantly higher yields of purified histones compared to crude acid extraction methods.

Total H3 Histone ELISA data for Histone Microplate Purification vs Crude Extraction
H3K9ac ELISA data for Histone Microplate Purification vs Crude Extraction
Figure 3: Determination of Histone H3 yields following Histone Microplate Purification.

Histones were extracted from 0.9 x 105 HeLa cells using the Histone Purification Microplate Kit. In the top image, the quantity of Total H3 recovered from the purification was determined using the Recombinant Histone H3 protein standard curve in the Total Histone H3 ELISA Kit (Catalog No. 53110) with 2 µl of purified histone sample or 200 ng crude acid extracted histone proteins. The Total H3 levels were consistent between both the untreated and treated HeLa cells as expected. The same samples were also run in the Histone H3K9ac ELISA (Catalog No. 53114) to show the increase in acetylation levels when the cells were treated with the HDAC inhibitor, sodium butyrate. In both assays, purified histones show greater sensitivity in the assay using less sample material than crude acid extracts.

Contents & Storage

The Histone Purification Microplate Kit contains sufficient reagents to perform 96 histone purifications.

The Histone Purification Microplate Kit contains reagents with multiple storage temperatures inside. Please confirm receipt of all reagents upon arrival and store items at the appropriate temperatures as indicated below. All reagents are guaranteed stable for 6 months from date of receipt when stored properly. This kit includes the following components:

  • Extraction Buffer (30 ml); Store at 4°C
  • 5X Neutralization Buffer (8 ml); Store at 4°C
  • Equilibration Buffer (60 ml); Store at 4°C
  • Histone Wash Buffer (60 ml); Store at 4°C
  • Histone Elution Buffer (60 ml); Store at 4°C
  • Purification Columns (96 ea); Store at RT
  • Collection/Elution Microplates (96 ea); Store at RT