Histone western blot

Histones are small proteins found in the nucleus that are crucial in packaging genomic DNA into structures called nucleosomes. Each nucleosome is created when about 146 base pairs of DNA wrap around a histone octamer, composed of two copies each of the core histones H2A, H2B, H3, and H4. The organization of nucleosome arrays into higher-order structures is facilitated by histone H1, which attaches to the linker DNA connecting neighboring nucleosomes.

Histones contain many positively charged amino acids, such as lysine (Lys) and arginine (Arg), enabling them to bind effectively to the negatively charged phosphate backbone of DNA. This close connection between nucleosomal DNA and histones restricts the access of transcription factors to the DNA. However, this access can be modified by post-translational modifications (PTMs) of the core histones.

The structures of the four core histones include central globular domains and long N-terminal tails, with the tails containing most of the amino acids that undergo PTMs. These modifications include various chemical changes to multiple amino acids, with acetylation and methylation of specific lysines being the most well-studied. These PTMs are frequently assessed both globally and at specific loci within the genome.

Histones and their PTMs are critical in regulating gene expression. Factors such as differentiation, environmental stressors, exposure to xenobiotics, and major human diseases can lead to significant changes in histone variants and their modifications. Western blotting is the primary method used in most studies to detect histones and their PTMs. This method allows researchers to separate histone proteins by size using gel electrophoresis. The proteins are then transferred onto a membrane, where specific histones or their modifications are detected using antibodies. This approach offers valuable insights into how histone modifications influence chromatin structure and function, ultimately affecting gene expression and cellular processes. By employing histone western blot analysis, scientists can investigate the dynamic changes in histone proteins that are fundamental to various aspects of cell biology and disease.

The following histone western blot protocol is routinely used at Abcam to detect histone proteins derived from purified calf thymus.
This protocol is widely used to detect histone modifications. Western blots can also detect other core histones and proteins present in chromatin preparations.
The small size of histones and other low molecular weight proteins can influence detection sensitivity and membrane choice during the separation and detection of histone proteins.
Signal visualization is achieved using appropriate detection reagents. Optimized protocols can result in strong signals for histone proteins and their modifications.

Procedure

Steps

For each lane, prepare 0.5 μg calf thymus or acid-extracted histones diluted in 1X LDS sample buffer supplemented with 100 mM DTT.

• Heat the sample to 95°C for 5 min.
• Centrifuge the sample briefly to restore the sample volume from condensation formed in the tube.

Prepare a 10% Bis-Tris gel, 1.0 mm thickness.

• A higher percentage gel (15%) is recommended for more effective resolution of histone proteins.

Load the histone samples, remembering to include a pre-stained protein standard.

• Run the gel in MES SDS running buffer at 200 V for 35 min.

For protein transfer from the gel, please refer to the protocols supplied with your transfer apparatus. These will vary depending upon the method of transfer employed, i.e., semi-dry blotting or wet blotting.

• Transfer times between 30 and 90 minutes should prove sufficient for effective protein transfer; we transferred at 30V for 70 min using 1X transfer buffer / 20% methanol.

Verify the successful transfer and equal loading of the histones using Ponceau staining.

• Dilute the Ponceau out of the membrane by adding dH₂O.

Block the membrane for 1 hr at room temperature (RT) using 5% BSA / 0.1% TBST (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20).

Cut the membrane into strips if necessary, and prepare the primary antibody by diluting it in blocking buffer (5% BSA / 0.1% TBST) at the dilution recommended by the Abcam datasheet.

• Add blocking peptides (at 1 μg/mL) as required and incubate on a rotating platform for 20 min at RT.
• Incubate the membrane with the primary antibody for 1.5 hr at RT or overnight at 4°C.

Rinse the blots briefly in 0.1% TBST and then perform two 5-minute washes followed by two 10-minute washes using the same buffer.

Incubate the membrane with the secondary antibody for 1 hr at RT [for example ab6721: Goat polyclonal to rabbit IgG H&L (HRP)] again diluted as recommended in 1% BSA / 0.1% TBST.

Wash the membrane in 0.1% TBST twice for 5 min, and twice for 10 min.

Perform ECL, ECF, or infrared detection as described by the manufacturer. For example, add ECL reagents for 3 min at RT and capture a WB image using various exposure durations: 10 sec, 30 sec, 1 min, 2 min, 3 min, 4 min, and 5 min.