Histone Extraction Kit

Synonyms

histone, extraction, isolation, acid precipitation, core histones, H2A, H2B, H3, H4, sample preparation, purify, post-translational modifications, acetylation, methylation, phosphorylation

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Overview

The Histone Extraction Kit provides a simple method to acid precipitate histones from cell culture, tissue and primary cells (e.g. T-cells) while preserving histone post-translational modifications (PTMs). The highly basic nature of histone proteins often precludes them from isolation using traditional methods for protein extraction. Instead, histones must be isolated under acidic conditions to retain solubility. The inclusion of inhibitors helps to preserve histone modifications such as acetylation, methylation and phosphorylation.

Each kit contains enough reagents for extractions from 2 x 96-well plates (192 rxns), or 100 rxns from cultured cell lines and tissues. Kits include buffers, protease inhibitors, phosphatase inhibitors, deacetylase inhibitors and optimized protocols for efficient recovery of core histones (H2A, H2B, H3 & H4).

Lysates prepare using the Histone Extraction Kit are compatible with downstream analysis by Western blot and Histone Modification ELISA. For researchers interested in a more purified histone lysate, Active Motif also offers Histone Purification Kits:

Description

Histone Extraction Kit Highlights

• Acid extraction protocol isolates highly basic histone proteins from cell culture, tissue and primary cells
• Kit includes protease inhibitors, phosphatase inhibitors and deacetylase inhibitors to preserve histone modifications
• Reagents provided for isolation from 2 x 96-well plates, or 100 cell culture or tissue extractions
• Compatible with downstream analysis by Western blot, ELISA or Active Motif's Histone H3 PTM Multiplex Kit

How does it work?

Active Motif's Histone Extraction Kit isolates core histone proteins (H2A, H2B, H3 & H4) from tissues and cell lines using an acid precipitation protocol that preserves histone post-translational modifications with the inclusion of phosphatase, protease and deacetylase inhibitors. For cultured cells either a one-step extraction or two-step extraction protocol can be used. Some cell lines are more resistant to cell lysis requiring the use of a two-step extraction method to first isolate the nuclei of the cells and then perform the histone extraction. Protocols are also provided to work with suspension cells, 96-well culture plates and tissue samples. A supplemental protocol is included when working with primary cells (e.g. T-cells). Core histones isolated by the Histone Extraction Kit can be used in downstream applications such as Western blot, Histone Modification ELISAs, or Active Motif's Histone H3 PTM Multiplex Kit.

Figure 1: SDS-PAGE and Western blot analysis of histone lysates.

A) SDS-PAGE gel using 20 µg mouse kidney histone lysate (lane 1) or 10 µg mouse kidney lysate (lane 2) shows histone lysate purity using the tissue extraction protocol in the Histone Extraction Kit. B) Western blot data using histone lysates isolated from untreated (-) and treated (+) HeLa cells that underwent a one-step extraction protocol. Twenty micrograms of each lysate was run per well and probed with a 1:500 dilution H3S10ph mAb (Cat. No. 39636), 1:250 dilution H3K27me3 pAb (Cat. No. 39155), 1:500 dilution H3K9ac mAb (Cat. No. 61251) and 1 µg/ml Histone H3 mAb (Cat. No. 61475). Results show the preservation of histone PTMs following extraction.

Input Requirements & Expected Yields

The amount of input material recommended per reaction will depend on the sample type:

• Cultured Cell Lines: 100,000 cells - 2 million cells per reaction
• 96-well culture plate: 5,000 cells - 50,000 cells per well
• Tissue samples: 5 mg - 50 mg per reaction

The yield will vary depending on the cell or tissue sample used. Some guidelines and expected yields are shown below:

Sample Types and Expected Yields

Sample Type	Input Amount	Protein Yield	Concentration (mg/ml)
Mouse Brain	51 mg	0.75 - 1.25 mg	3.0 - 4.5 mg/ml
Mouse Lung	62 mg	0.6 - 0.75 mg	2.0 - 3.0 mg/ml
Mouse Kidney	51 mg	0.6 - 0.9 mg	2.4 - 3.7 mg/ml
Mouse Spleen	58 mg	1.4 - 1.6 mg	2.4 - 5.8 mg/ml
Mouse Liver	58 mg	1.5 - 2.0 mg	6.5 - 7.6 mg/ml
Mouse Heart	52 mg	0.75 - 1.0 mg	3.5 - 4.0 mg/ml
Mouse Colon	52 mg	0.75 - 1.0 mg	2.5 - 3.5 mg/ml
6-well plate	300,000 cells	~220 µg	not available
12-well plate	100,000 cells	~75 µg	not available
24-well plate	50,000 cells	~37.5 µg	not available

Contents

Contents & Storage

The Histone Extraction Kit contains sufficient reagents to perform 192 histone extractions from 2 x 96-well plates or 100 extractions from cultured cells or tissues.

The Histone Extraction 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:

• Neutralization Buffer AM2; Store at RT
• 10X PBS; Store at 4°C
• Lysis Buffer AM8; Store at 4°C
• Extraction Buffer; Store at 4°C
• Deacetylase Inhibitor; Store at -20°C
• Protease Inhibitor Cocktail (PIC); Store at -20°C
• Phosphatase Inhibitors; Store at RT to -20°C
• 1 M DTT; Store at -20°C

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