Histone western blot

​​​Kindly provided by Prof. Tony Kouzarides, University of Cambridge.

Last edited Wed 01 Feb 2023

The following histone western blot protocol is routinely used at abcam for the detection of histone proteins derived from purified calf thymus.

Stage 1 - 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.

Please note that protein loadings derived from cellular lysates will need to be determined empirically.

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

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

It is advisable not to run the dye front completely off the gel when dealing with smaller protein resolutions.

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

Nitrocellulose membranes with a pore size of 0.2 mm are recommended for optimal retention of histone proteins.

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

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 in blocking buffer (5% BSA / 0.1% TBST) at a dilution recommended by the Abcam datasheet.

Rinse the blots briefly in 0.1% TBST and then perform two 5 min washes followed by two 10 min 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, e.g. add ECL reagents for 3 min at RT and capture WB image using various durations of exposure: 10 sec, 30 sec, 1 min, 2 min, 3 min, 4 min and 5 min.