Home › Neuroscience › Purification of H3 and H4 Histone Proteins and the Quantification of Acetylated Histone Marks in Cells and Brain Tissue
Neuroscience JoVE (Open Access) Citable · DOI

Purification of H3 and H4 Histone Proteins and the Quantification of Acetylated Histone Marks in Cells and Brain Tissue

DOI: 10.3791/58648-v
What you'll learn
  • ✓ Purify core histone proteins H3 and H4 from cells and tissue samples
  • ✓ Quantify acetylated histone residues using biochemical methods
  • ✓ Evaluate histone purity and identify non-histone protein contamination
  • ✓ Apply histone extraction protocols to cultured cells and brain tissue
Protocol

Biopharma Insights The purpose of this article is to provide a comprehensive, systematic guide to the efficient purification of histones H3 and H4 and the quantification of acetylated histone residues.

Difficulty
advanced
Total time
~4-6 hours per sample batch (including precipitation and purification steps)
Model organism
Mouse (brain tissue); BV2 microglial cells (cultured)
Biosafety
BSL-1

Steps

1
Prepare sample extract from cells or tissue

Lyse cells or homogenize tissue samples to create a crude cellular extract. This extract serves as the starting material for histone purification.

▶ 01:06
2
Generate crude histone extract from lysate

Process the cell lysate through acid extraction and centrifugation steps to enrich histone proteins while removing most non-histone proteins.

▶ 02:38
3
Evaluate histone and non-histone protein presence

Analyze the crude extract using gel electrophoresis or biochemical assays to confirm histone enrichment and assess contamination levels.

▶ 03:33
4
Purify core histones H3 and H4

Apply chromatography or selective extraction methods to isolate and separate core histone proteins from the crude extract.

▶ 04:14
5
Precipitate and collect purified core histones

Use precipitation methods to concentrate and isolate the purified histone proteins for downstream quantification and analysis.

▶ 05:13
6
Quantify acetylated histone marks in samples

Apply immunological or mass spectrometry-based methods to quantify acetylation status of histone residues in purified H3 and H4 from cultured cells and brain tissue.

▶ 07:02
💬 Comments coming soon