实验概要

The method provides a procedure and tips for detecting histone proteins.The protocol refers to the western blot detection of histone proteins derived from purified calf thymus performed at Abcam.

实验步骤

1. 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.

2. Prepare a 10% NuPAGE Bis Tris gel 1.0 mm. A higher percentage gel (15%) is recommended for more effective resolution of histone proteins.

3. 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.

4. 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.

5. Verify the successful transfer and equal loading of the histones using Ponceau staining. Dilute the Ponceau out of the membrane by adding dH2O.

6. 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).

7. 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.

8. 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.

9. 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)).

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

11. 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.