The following protocol refers to the western blot detection of histone proteins derived from purified calf thymus performed at Abcam.
- For each lane prepare 0.5 μg calf thymus or acid extracted histones diluted in 1X NuPAGE LDS sample buffer (Invitrogen) supplemented with 100 mM DTT. Heat the sample to 95°C for 5 minutes. Centrifuge the sample briefly to restore sample volume from condensation formed in the tube during heating.
- Prepare a 10% NuPAGE Bis Tris gel 1.0 mm. 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 (Precision Plus Protein Standard (Kaleidoscope), Bio-Rad). Run the gel in NuPAGE MES SDS running buffer at 200 V for 35 mins.
NB It is advisable not to run the dye front completely off the gel.
- Transfer the protein samples onto a nitrocellulose membrane with reduced pore sizes (Invitrogen; LC2000) at 30 V for 70 minutes using NuPAGE transfer buffer (1X) / 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 hour 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. Add blocking peptides as required and incubate on a rotating platform for 20 minutes at RT. Incubate the membrane with the primary antibody for 1.5 hours 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 hour at RT, diluted in 5% BSA / 0.1% TBST. (For example ab6721: Goat polyclonal to rabbit IgG H&L (HRP)).
- Wash the membrane in 0.1% TBST twice for 5 minutes, and twice for 10 minutes.
- Add ECL reagents for 3 minutes at RT. Capture WB image using Syngene GeneGnome using various durations of exposure: 10 s, 30 s, 1 min, 2 min, 3 min, 4 min and 5 min.
Top tips for successful western blotting with our range of histone antibodies.
Use a high percentage gel for clear resolution of histone proteins.